Extensive Cross-reactivity Research Articles

A Phl p 7-specific IgG4 antibody inhibits allergic patients IgE cross-reactivity to allergens from the EF-hand family: importance of affinity and degree of cross-reactivity

Methods and results Sequence comparison showed a comparable sequence identity of approximately 70% between the 2EF-hand pollen allergens (Timothy grass: Phl p 7; Alder: Aln g 4; Birch: Bet v 4; Turnip rape: Bra r 1; Lamb s quarter: Che a 3; Olive: Ole e 3) and a lower sequence identity of 44% with the 4EF-hand allergen from olive pollen (Ole e 8). While patients IgE showed extensive cross-reactivity with the EF-hand allergens, the Phl p 7-specific IgG4 showed limited cross-reactivity. Best cross-reactivity was observed with Ole e 3 whereas the other EF-hand allergens were less recognized by the Phl p 7-specific IgG4 in dot blot experiments. Calcium-depletion experiments showed that the binding of the Phl p 7-specific IgG4 depended on the presence of calcium. Using peptidespecific antisera the binding site of the IgG4 was located in the C-terminal portion of the allergen which contains the second calcium-binding EF-hand motif. Biacore experiments revealed interesting differences regarding the affinity of the Phl p 7-specific IgG4. Phl p 7 and Ole e 3 were recognized with the highest affinity (KD s: Phl p 7: 2,11x10-9M; Ole e 3: 6,18x10-9M) whereas the affinities to the other cross-reactive allergens was much lower (Bet v 4: 6,26x10-6M; Bra r 1: 6,57x10-6M; Aln g 4: 7,93x10-6M). The cross-inhibition of patients IgE reactivity to the EF-hand allergens and the inhibition of allergen-induced basophil activation obtained with the Phl p 7-specific IgG4 followed its intensity of cross-reactivity and affinity with the homologous allergens.

Papulovesikuläres Exanthem nach einer Südafrika-Safari

A 68-year-old man presented with a papulovesicular exanthem, fever and malaise after a safari in South Africa. Based on the history, the typical clinical picture with an exanthema and eschar as well as the detection of antibodies against rickettsioses of the spotted fever group, we diagnosed African tick-bite fever which is due to R.africae. During treatment with doxycycline 200mg/d, all symptoms resolved completely within 11 days. Rickettsioses should always be considered in patients presenting with exanthema, fever and malaise. Particularly the presence of one or multiple eschars on the skin manifesting as erythematous plaques with central necrosis is a pathognomic sign. The serological detection of antibodies against rickettsia species of the spotted fever group is the established diagnostic standard. Due to extensive cross-reactions it is not possible to distinguish between the members of one rickettsial group. Furthermore antibody titers rise late in the disease, frequently 2 or 3 weeks after the onset of symptoms. This underscores the importance of the clinical diagnosis.

LC–MS/MS in Endocrinology: What is The Profit of The Last 5 Years?

Currently, chromatography (GC but more commonly HPLC) is the analytical method of choice for several hormones, either because the immunoassays suffer from extensive crossreactivity or because chromatography permits simultaneous measurements of hormones. However, sometimes the conventional detection systems with HPLC methods do not meet desired specificity. With the increase of robust and affordable LC-MS/MS systems, the next step forward in specificity was taken. LC-MS/MS is rapidly being incorporated in the endocrine laboratories. To be useful in the clinical diagnostic practice, it is of utmost importance that methods are both analytically and clinically vaidated, as until now, the majority of applications of LC-MS/MS in the clinical laboratories are 'home-made' methods, therefore special case must be taken. This review aims to focus on Clinical and Laboratory Standards Institute or comparable validated LC-MS/MS methods for targeted hormone analysis used for diagnostic purposes in human samples, published in the last 5 years.

Determination of zeranol and its metabolites in bovine muscle and liver by a chemiluminescence enzyme immunoassay: compared to an ultraperformance liquid chromatography tandem mass spectroscopy method

A chemiluminescent enzyme immunoassay (CLEIA) was compared to an ultraperformance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) procedure for the analysis of zeranol and its metabolites in bovine tissue samples. Apparent recoveries from fortified samples by both methods were comparable at 0.5-4.0 µg/kg and a significant correlation was obtained. For CLEIA analysis, hapten mimicking the analyte was first synthesized and conjugated with the carrier protein bovine serum albumin as the immunogen to produce monoclonal antibody. The obtained antibody showed extensive cross-reactivity toward zeranol metabolites (zearalanone). The limit of detection of CLEIA and UPLC-MS/MS was 0.05 µg/kg and 0.5 µg/kg, respectively. Recoveries of both methods for fortified samples were higher than 75.0% with the coefficient of variation less than 15%. These results indicated that the combination of screening with CLEIA and confirmation with UPLC-MS/MS for zeranol and its metabolites would be a reliable method for a large number of bovine samples.

Cross-reacting carbohydrate determinants and hymenoptera venom allergy

Insect venom allergy is an important cause of anaphylaxis. Venom immunotherapy assume the clear identification of the culprit insect, but this is impeded by Immunoglobulin E (IgE) antibodies to cross reactive carbohydrate determinant (CCD) epitopes of common glycoproteins. Here we give an overview about inducers, importance, and relevance of anti-N-Glycan CCD IgE antibodies. Pollen exposure and insect stings induce anti-CCD IgE antibodies interfering with in-vitro tests for allergy diagnosis due to extensive IgE cross-reactivity. Instead of being biologically active these antibodies are irrelevant for allergic reactions due to hymenoptera stings. The general response of the immune system to the ubiquitous exposure to N-glycan containing glycoproteins is still a matter of debate. CCD specific IgG antibodies in sera of bee keepers suggest tolerance induction due to high-dose exposure. Tolerance induction by pollen and food glycoproteins has not been proved. Hymenoptera stings and pollen exposure induce anti-CCD IgE. In regard to anaphylaxis due to Hymenoptera stings these antibodies are not clinically relevant, but they are important for the specificity of in-vitro tests proving insect venom allergy. The introduction of component based diagnostic IgE testing improves the specificity of in-vitro tests if proteins devoid of CCD epitopes are used.

Neurofascin 186 specific autoantibodies induce axonal injury and exacerbate disease severity in experimental autoimmune encephalomyelitis

Axonal injury is considered the major cause of chronic disability in multiple sclerosis (MS) patients, however the mechanisms behind remain still unclear. Recently, it was demonstrated that autoantibodies against Neurofascin, a cell adhesion molecule within the adult nervous system, can contribute to the development of axonal pathology in some patients.We compared the ability of the two different isoforms of Neurofascin, Nfasc155 and Nfasc186, to induce a pathogenic antibody response in the Dark Agouti (DA) rat. Animals were immunized with recombinant proteins prior to induction of experimental autoimmune encephalomyelitis (EAE) by adoptive transfer of activated MOG-specific T cells. Only Nfasc186 induced an axopathic autoantibody response in vivo, despite extensive cross reactivity between the two isoforms as shown by ELISA and flow cytometry. In this case, using transfected cell lines failed to differentiate between pathogenic and non-pathogenic responses. These findings have important implications with respect to the usage of cell based assays as an approach to detect pathologically relevant autoantibodies in clinical samples.

Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy

The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy.Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE.Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.

The two-faced T cell epitope

Advances in the field of T cell immunology have contributed to the understanding that cross-reactivity is an intrinsic characteristic of the T cell receptor (TCR), and that each TCR can potentially interact with many different T cell epitopes. To better define the potential for TCR cross-reactivity between epitopes derived from the human genome, the human microbiome, and human pathogens, we developed a new immunoinformatics tool, JanusMatrix, that represents an extension of the validated T cell epitope mapping tool, EpiMatrix. Initial explorations, summarized in this synopsis, have uncovered what appear to be important differences in the TCR cross-reactivity of selected regulatory and effector T cell epitopes with other epitopes in the human genome, human microbiome, and selected human pathogens. In addition to exploring the T cell epitope relationships between human self, commensal and pathogen, JanusMatrix may also be useful to explore some aspects of heterologous immunity and to examine T cell epitope relatedness between pathogens to which humans are exposed (Dengue serotypes, or HCV and Influenza, for example). In Hand-Foot-Mouth disease (HFMD) for example, extensive enterovirus and human microbiome cross-reactivity (and limited cross-reactivity with the human genome) seemingly predicts immunodominance. In contrast, more extensive cross-reactivity with proteins contained in the human genome as compared to the human microbiome was observed for selected Treg epitopes. While it may be impossible to predict all immune response influences, the availability of sequence data from the human genome, the human microbiome, and an array of human pathogens and vaccines has made computationally–driven exploration of the effects of T cell epitope cross-reactivity now possible. This is the first description of JanusMatrix, an algorithm that assesses TCR cross-reactivity that may contribute to a means of predicting the phenotype of T cells responding to selected T cell epitopes. Whether used for explorations of T cell phenotype or for evaluating cross-conservation between related viral strains at the TCR face of viral epitopes, further JanusMatrix studies may contribute to developing safer, more effective vaccines.

Advances in T-Cell Epitope Engineering

T-cells recognize small peptide fragments (p) cradled in multiple major histocompatibility complex (MHC) molecules, termed human leukocyte antigens (HLA) in humans. These membrane-integral pMHC molecules are present on surface of all nucleated cells and allow T-cells to detect aberrant intracellular activity, be this infection with microorganisms or abnormal host biochemistry such as neoplastic division. Scanning of pMHC molecules occurs via the αβ T-cell receptor (TCR), a clonotypic, heterodimeric, and membrane-integral molecule on the T-cell surface (Miles et al., 2011a). TCRs engage pMHC molecules via six highly flexible complementarity determining region (CDR) loops and, upon productive docking with a pMHC molecule, the TCR triggers a myriad of intracellular T-cell signaling cascades (Bridgeman et al., 2012). The binding strength (or affinity) between a TCR and a cognate pMHC is relatively weak across known biological systems with monomeric “dwell times” (or half-lives) typically measured in seconds or microseconds at physiological temperatures (Miles et al., 2010; Bridgeman et al., 2012; Smith et al., 2012). This is in contrast to numerous other biological interactions such as antibodies (van der Merwe and Davis, 2003), interleukins (Morton et al., 1994), lipoproteins (Misra et al., 2001), and structural membrane proteins (Matte et al., 2012) which have half-lives measured in hours-to-days. Overall, TCR/pMHC interactions are fleeting even by the dynamic standards of cell surface interactions (van der Merwe and Davis, 2003). The evolutionary rationale for this striking functional divide can only be speculated upon but likely pertains to the primary function of T-cells. T-cells must scan large numbers of pMHC on multiple cells in series in order to identify and eliminate threats quickly. Effective immunity requires that TCR scanning time must be minimal and antigen coverage maximal. Theoretical arguments dictate that maximal immune cover of possible foreign pMHC requires each TCR to recognize huge numbers of different peptides (Mason, 1998; Sewell, 2012). This theory is now supported up by direct experimental evidence that shows a single TCR can cross-recognize millions of pMHC molecules as well or better than the native antigen (Sewell, 2012; Wooldridge et al., 2012; Ekeruche-Makinde et al., 2013). Curiously, this extensive T-cell cross-reactivity is strictly compartmentalized based on peptide length (Ekeruche-Makinde et al., 2013).

HAZELNUT ALLERGY: A MULTI-FACED CONDITION WITH DEMOGRAPHIC AND GEOGRAPHIC CHARACTERISTICS

Hazelnut (Corylus avellana) allergy varies from rather mild oral allergy symptoms to potentially life-threatening anaphylaxis and exhibits geographic and age-related variations. Severity of symptoms depends on the sensitisation profile of the patient and can partially be predicted using ‘component-resolved diagnosis’. In our region (young) children predominantly exhibit sensitisation to hazelnut storage proteins Cor a 9 and Cor a 11 that is unrelated to birch pollen allergy and is generally associated with a more severe clinical outcome on consumption on raw and processed hazelnut. In contrast, adults predominantly present with an oral allergy syndrome due to an extensive cross-reactivity between the labile Cor a 1.04 and Bet v 1, the major allergen from birch (Betula verrucosa) pollen. In the absence of a cure, avoidance remains the key measure of effective management, particularly in those patients presenting with a severe form.

Cross‐allergic Reactions to Legumes in Lupin and Fenugreek‐Sensitized Mice

Several legumes may induce allergy, and there is extensive serological cross-reactivity among legumes. This cross-reactivity has traditionally been regarded to have limited clinical relevance. However, the introduction of novel legumes to Western countries may have changed this pattern, and in some studies cross-allergy to lupin has been reported in more than 60% of peanut-allergic patients. We wanted to explore cross-reactions among legumes using two newly established mouse models of food allergy. Mice were immunized perorally with fenugreek or lupin with cholera toxin as adjuvant. The mice were challenged with high doses of fenugreek, lupin, peanut or soy, and signs of anaphylactic reactions were observed. Cross-allergic mechanisms were investigated using serum mouse mast cell protease-1 (MMCP-1), antibody responses, immunoblotting and ex vivo production of cytokines by spleen cells. Signs of cross-allergy were observed for all the tested legumes in both models. The cross-allergic symptoms were milder and affected fewer mice than the primary allergic responses. The cross-allergy was reflected to a certain extent in the antibody and T-cell responses, but not in serum MMCP-1 levels. Cross-allergy to peanut, soy, fenugreek and lupin was observed in lupin-sensitized and fenugreek-sensitized mice. Differences in serological responses between primary allergy and cross-allergy might be due to mediation through different immune mechanisms or reflect different epitope affinity to IgE. These differences need to be further investigated.

Abstract 4067: Integrated microfluidic bead-based immunosensor for multiplexed determinations of ovarian cancer biomarkers at the point-of-care

Abstract Early detection of ovarian cancer could significantly impact clinical outcome and hence patient survival. Given the biological heterogeneity of ovarian cancer, multi-marker panels will be required. Validation of such panels must utilize pre-clinical serum samples that are available only in small quantities. Diagnostic platforms that are capable of rapid, precise and multiplexed analysis of biomarkers in small quantities of serum should facilitate validation of optimal biomarker panels. Once validated, these platforms could permit rapid screening with finger-stick quantities of blood at point-of-care. The Programmable Bio-Nano-Chip (p-BNC) microfluidic immunosensor provides such a platform. In the p-BNC, serum biomarkers are sequestered and assessed with a fluorescence-based sandwich immunoassay, completed in nano-nets of sensitized agarose microbeads localized in individually addressable wells, housed in a microfluidic module, capable of integrating multiple sample, reagent and biowaste processing and handling steps. Previously, we adapted the p-BNC for the quantification of the CA125 biomarker with low LODs, high precision and a short analysis time of 43 minutes. Here, we adapt the p-BNC to simultaneously assess CA125 and HE4, a promising biomarker panel implicated in early detection. The HE4 immunoassay was developed to perform optimally with the previously established CA125 assay as the rate determining step. Extensive cross reactivity analysis ruled out non-ideal interactions between HE4 and CA125 reagents at the level of capturing antibody, detecting antibody and individual analytes. Wash times were optimized to maximize p-BNC performance. The dose-response curves obtained for the individual analytes (singleplex) were similar to those obtained with multiplex assays. Identical LOD values were noted for the singleplex and multiplex immunoassays with 1.5-fold decrease in precision for the multiplexed assays, demonstrating negligible loss of analytical performance upon multiplexing. For clinical validation, 8 surgically confirmed advanced stage patient sera and 8 age-matched healthy controls were assessed on the multiplexed p-BNC. ROC curve analysis for the CA125-HE4 biomarker combination, interpreted with a Risk of Ovarian Malignancy Algorithm was able to discriminate diseased samples from healthy controls with an AUC of 1.00. The p-BNC indicates strong promise for robust, multiplexed, quantitative measurements of ovarian cancer biomarkers, with high precision and sensitivity to yield clinically pertinent information under 45 minutes. Incorporation of additional biomarkers is underway to establish and validate an optimal panel for early detection of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4067. doi:1538-7445.AM2012-4067

West Nile virus: characterization and diagnostic applications of monoclonal antibodies

BackgroundDiagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs.MethodsSix MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity.ResultsAll MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys → Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser → Ile) or E278 (Thr → Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs.ConclusionsMAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization.

Characterisation of vaccine-induced, broadly cross-reactive IFN-γ secreting T cell responses that correlate with rapid protection against classical swine fever virus

Live attenuated C-strain classical swine fever viruses (CSFV) provide a rapid onset of protection, but the lack of a serological test that can differentiate vaccinated from infected animals limits their application in CSF outbreaks. Since immunity may precede antibody responses, we examined the kinetics and specificity of peripheral blood T cell responses from pigs vaccinated with a C-strain vaccine and challenged after five days with a genotypically divergent CSFV isolate. Vaccinated animals displayed virus-specific IFN-γ responses from day 3 post-challenge, whereas, unvaccinated challenge control animals failed to mount a detectable response. Both CD4+ and cytotoxic CD8+ T cells were identified as the cellular source of IFN-γ. IFN-γ responses showed extensive cross-reactivity when T cells were stimulated with CSFV isolates spanning the major genotypes. To determine the specificity of these responses, T cells were stimulated with recombinant CSFV proteins and a proteome-wide peptide library from a related virus, BVDV. Major cross-reactive peptides were mapped on the E2 and NS3 proteins. Finally, IFN-γ was shown to exert potent antiviral effects on CSFV in vitro. These data support the involvement of broadly cross-reactive T cell IFN-γ responses in the rapid protection conferred by the C-strain vaccine and this information should aid the development of the next generation of CSFV vaccines.

A Diagnostic Algorithm to Serologically Differentiate West Nile Virus from Japanese Encephalitis Virus Infections and Its Validation in Field Surveillance of Poultry and Horses

The detection of West Nile virus (WNV) in areas endemic for Japanese encephalitis virus (JEV) is complicated by the extensive serological cross-reactivity between the two viruses. A testing algorithm was developed and employed for the detection of anti-WNV antibody in areas endemic for JEV. Using this differentiation algorithm, a serological survey of poultry (2004 through 2009) and horses (2007 through 2009) was performed. Among 2681 poultry sera, 125 samples were interpreted as being positive for antibodies against JEV, and 14 were suspected to be positive for antibodies against undetermined flaviviruses other than WNV and JEV. Of the 2601 horse sera tested, a total of 1914 (73.6%) were positive to the initial screening test. Of these positive sera, 132 sera (5.1%) had been collected from horses that had been imported from the United States, where WNV is endemic. These horses had WNV vaccination records, and no significant pattern of increasing titer was observed in paired sera tests. Of the remaining 1782 positive sera 1468 sera (56.4%) were also found to contain anti-JEV antibodies, and were interpreted to be JEV-specific antibodies by the differentiation algorithm developed in this study. The remaining 314 horses (12.1%) for which a fourfold difference in neutralizing antibody titer could not be demonstrated, were determined to contain an antibody against an unknown (unidentified or undetermined) flavivirus. No evidence of WNV infections were found during the period of this study.

Optimization of Peptide-Based ELISA for Serological Diagnostics: A Retrospective Study of Human Monkeypox Infection

Although smallpox has been eradicated, other diseases caused by virulent orthopoxviruses such as monkeypox virus (MPV) remain endemic in remote areas of western and central sub-Saharan Africa, and represent a potential biothreat due to international travel and/or inadvertent exposure. Unfortunately, extensive antigenic cross-reactivity among orthopoxviruses presents a challenge to serological diagnosis. We previously reported a 20mer peptide-based ELISA that identified recent MPV infection with >90% sensitivity and >90% specificity. However, the sensitivity of this approach was not determined with samples obtained at later time points after antibody titers had declined from their peak levels. To improve assay sensitivity for detecting MPV-specific antibodies at later time points, we compared diagnostic 20mer peptides to 30mer peptides. In addition, optimal 30mer peptides were tested in combination or after conjugating selected peptides to a carrier protein (bovine serum albumin) to further improve assay performance. An optimized combination of four unconjugated 30mer peptides provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 45% sensitivity for detecting MPV infection at >2 years post-infection, and 99% specificity. However, an optimized combination of two peptide conjugates provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 90% sensitivity for detecting MPV infection at >2 years post-infection, and 97% specificity. Peptide-based ELISA tests provide a relatively simple approach for serological detection of MPV infection. Moreover, the systematic approach used here to optimize diagnostic peptide reagents is applicable to developing improved diagnostics to a broad range of other viruses, and may be particularly useful for distinguishing between closely-related viruses within the same genus or family.

Kidney Bean: A Major Sensitizer among Legumes in Asthma and Rhinitis Patients from India

BackgroundThe prevalence of IgE mediated food allergies has increased over the last two decades. Food allergy has been reported to be fatal in highly sensitive individuals. Legumes are important food allergens but their prevalence may vary among different populations. The present study identifies sensitization to common legumes among Indian population, characterizes allergens of kidney bean and establishes its cross reactivity with other legumes.MethodologyPatients (n = 355) with history of legume allergy were skin prick tested (SPT) with 10 legumes. Specific IgE (sIgE) and total IgE were estimated in sera by enzyme-linked immunosorbent assay. Characterization of kidney bean allergens and their cross reactivity was investigated by immunobiochemical methods. Identification of major allergens of kidney bean was carried out by mass spectrometry.Principal FindingsKidney bean exhibited sensitization in 78 (22.0%) patients followed by chickpea 65 (18.0%) and peanut 53 (15%). SPT positive patients depicted significantly elevated sIgE levels against different legumes (r = 0.85, p<0.0001). Sera from 30 kidney bean sensitive individuals exhibited basophil histamine release (16–54%) which significantly correlated with their SPT (r = 0.83, p<0.0001) and sIgE (r = 0.99, p<0.0001). Kidney bean showed eight major allergens of 58, 50, 45, 42, 40, 37, 34 and 18 kDa on immunoblot and required 67.3±2.51 ng of homologous protein for 50% IgE inhibition. Inhibition assays revealed extensive cross reactivity among kidney bean, peanut, black gram and pigeon pea. nLC-MS/MS analysis identified four allergens of kidney bean showing significant matches with known proteins namely lectin (phytohemagglutinin), phaseolin, alpha-amylase inhibitor precursor and group 3 late embryogenesis abundant protein.Conclusion/SignificanceAmong legumes, kidney bean followed by chick pea and peanut are the major allergic triggers in asthma and rhinitis patients in India. Kidney bean showed eight major allergens and cross reacted with other legumes. A combination of SPT, sIgE and histamine release assay is helpful in allergy diagnosis.

Cold-Adapted Influenza and Recombinant Adenovirus Vaccines Induce Cross-Protective Immunity against pH1N1 Challenge in Mice

BackgroundThe rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1) highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus.Methodology/Principal FindingsBALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca) influenza viruses from 1977 or recombinant adenoviruses (rAd) expressing 1934 nucleoprotein (NP) and consensus matrix 2 (M2) (NP+M2-rAd). Antibodies against the M2 ectodomain (M2e) were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus.Conclusion/SignificanceCross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic.

Extensive Horizontal Gene Transfer in Ureaplasmas from Humans Questions the Utility of Serotyping for Diagnostic Purposes

Ureaplasma parvum and Ureaplasma urealyticum are sexually transmitted, opportunistic pathogens of the human urogenital tract. There are 14 known serovars distributed between the two species. For decades, it has been postulated based upon limited data that virulence is related to serotype specificity. The results were often inconclusive due to the small sample size and extensive cross-reactivity between certain serovars. We developed real-time quantitative PCRs that allow reliable differentiation of the two species and type strains of each of the 14 serovars. To investigate species and serovar distributions, we typed 1,061 clinical isolates of human ureaplasmas from diverse patient populations. There was only a tenuous association between individual Ureaplasma serovars and certain patient populations. This may in part be explained by the fact that almost 40% of the isolates were genetic mosaics, apparently arising from the recombination of multiple serovars. This explains the extensive cross-reactivity based upon serotyping and the lack of consistent association of given serotypes with disease.

Immunochemical Approach Using Monoclonal Antibody against &amp;#x394; 9-Tetrahydrocannabinolic Acid (THCA) to Discern Cannabis Plants and to Investigate New Drug Candidates

A monoclonal antibody (MAb-4A4) against Δ9- tetrahydrocannabinolic acid (THCA) showing extensive cross-reactivity against various cannabinoids was prepared. Using this antibody, a competitive enzyme-linked immunoassay (ELISA) was developed to detect Δ9-THCA in the range of 1 to 100 mg/ml. Various cannabinoids including Δ9-THC (Δ9-tetrahydrocannabinolic acid), Δ8-THCA (Δ8-tetrahydrocannabinolic acid), Δ8-THC (Δ8-tetrahydrocannabinol), CBD (cannabidiol), and CBN (cannabinol) were recognized by MAb-4A4, and their cross-reactivities were 55-1600% compared with Δ9-THCA (100%). This novel characteristic of this MAb enabled detection of marijuana residues in biological samples by detection of residual cannabinoids. The ELISA using MAb-4A4 was found to be applicable even for withered samples which contained only trace amounts of Δ9-THCA and Δ9-THC. In addition, this method using MAb-4A4 could be useful in forensic analysis since the MAb-4A4 also shows cross-reactivities against cannabinoid metabolites in body fluids. As well as forensic applications using this MAb, an investigation of new drug candidates focusing on cannabinoid metabolites arising from biotransformation in plant tissue was performed using immunochemical screening. The resulting new drug candidates were cannabinoid glycosides biotransformed by Pinellia ternata whose bioactivity is as yet unidentified. Our results indicate the utility of the application of ELISA using MAb-4A4 for further experiments involving marijuana and cannabinoids not only in the forensic field but also in the context of drug discovery.