In silico food allergenic risk evaluation of proteins extracted from macroalgae Ulva sp. with pulsed electric fields.

Extraction of protein from macroalgae, currently defined as "novel food", is challenging and limited information about the health impacts of these proteins is available. Here, we report on a non-thermal, chemical-free green macroalgae Ulva sp. protein extraction by osmotic shock combined with pulsed electric fields (PEF) followed by hydraulic pressure. The extracted proteins were identified and annotated to allergens using sequence similarity. The allergenicity potential of PEF extracted proteins was compared to osmotic shock extracts and complete Ulva sp. proteome, extracted with the thermochemical method. The PEF extracts contained 'superoxide dismutase' (SOD), a known food allergen, osmotic shock extract contained 'troponin C', and thermochemical extract contained two additional potential food allergens 'aldolase A' and 'thioredoxin h'. This study shows an importance and the need for deep investigation of algal proteins and protein extraction technology health impacts prior to large-scale release to the market of "novel food" derived proteins.

A smart nanosensor for the detection of human immunodeficiency virus and associated cardiovascular and arthritis diseases using functionalized graphene-based transistors.

Human immunodeficiency virus (HIV), which isa worldwide public health issue, is commonly associated with cardiovascular disorders (CVDs) and rheumatoid arthritis (RA). A smart nanosensor was developed for the detection of HIV and its related diseases (CVDs and RA) using graphene-based field-effect transistors (FETs). In this study, amine-functionalized graphene (afG) was conjugated with antibodies [anti-p24 for HIV, anti-cardiac troponin 1 (anti-cTn1) for CVDs, and anti-cyclic citrullinated peptide (anti-CCP) for RA] to detect various biomarkers. The antibodies were covalently conjugated to afG via carbodiimide activation. The bioconjugate (graphene-antibody) was characterized by various biophysical techniques such as UV-Vis, Raman spectroscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). The electrochemical performance of the sensor was evaluated with respect to changes in the resistance of the electrode surface due to the interaction of the antigen with its specific antibody. The developed sensor was highly sensitive and showed a linear response to p24, cTn1, and, CCP from 1 fg/mL to 1 µg/mL. The limit of detection (LOD) was 100 fg/mL for p24 and 10 fg/mL for cTn1 and CCP under standard optimized conditions. The graphene-based smart nanodevice demonstrated excellent performance; thus, it could be used for the on-site detection of HIV, CVD, and RA biomarkers in real samples.

Evaluating troponin C from Psoroptes cuniculi as a diagnostic antigen for a dot-ELISA assay to diagnose mite infestations in rabbits.

The mite Psoroptes cuniculi is globally widespread and has a serious impact on commercial rabbit breeding. In China, diagnosis of P. cuniculi is currently based on conventional clinical methods that entail numerous disadvantages, including their failure to diagnose subclinical infections. Hence, alternative measures are required, and dot-ELISA is one of the most promising strategies. We cloned and expressed the recombinant P. cuniculi troponin C gene for use as a basis for novel dot-ELISA assay to detect P. cuniculi infections in rabbits. This amplified sequence encoded a 153 amino acid protein of 17·6 kDa and theoretical pI 4·18 without signal peptide. The recombinant troponin C of P. cuniculi is an outer membrane protein and may also be a new P. cuniculi allergen. Results of dot-ELISA test showed that this novel assay had more than 90% sensitivity but low specificity in distinguishing infections with P. cuniculi or Sarcoptic scabiei, despite very high agreement between observers (97-99%; κ values ranged from 0·95 to 0·98 for inter- and intra-observer variability test). This study showed that this novel method, at present, lacks diagnostic utility. Therefore, although simple serological assays such as dot-ELISA show great promise as diagnostic tools, we suggest that troponin C is not a suitable diagnostic antigen candidate.

Allergenicity of recombinant troponin C from Tyrophagus putrescentiae.

BACKGROUND: The storage mite, Tyrophagus putrescentiae, produces potent allergens, many of which have not been characterized. This study was undertaken to characterize the allergenicity of troponin C from T. putrescentiae. METHODS: A cDNA encoding 17.7 kDa troponin C, with homology to cockroach allergen Bla g 6, was identified from T. putrescentiae-expressed sequence tags. Recombinant troponin C was expressed and IgE responses to the recombinant protein were assessed in the presence and absence of 10 mM CaCl(2). Cross-reactivity between T. putrescentiae troponin C and Bla g 6 was tested using an inhibition ELISA. RESULTS: Recombinant T. putrescentiae troponin C shares 62.7-85.5% homology with troponin C from various arthropods. Sera from 5 of 47 subjects in our study group (10.6%) showed IgE binding to the recombinant protein. Interestingly, addition of 10 mM CaCl(2) increased the intensity of IgE binding approximately 2-fold. In an immune-inhibition ELISA with these sera, T. putrescetiae troponin C and Bla g 6 did not cross-react significantly. CONCLUSIONS: Troponin C is a new mite allergen with calcium-dependent IgE reactivity.

Assessment of the interaction between a synthetic epitope of troponin C and its specific antibody using a label-free faradaic impedimetric immunosensor and alpha-Keggin silicotungstic heteropolyacid as a redox probe.

The development of an immunosensor for the direct probing of the interaction between a cysteine-modified synthetic peptide, which corresponds to the epitope cTnC-89-98 of troponin C, and its specific antibody is described. Following immobilization of the peptide onto gold electrodes through the formation of a self-assembled monolayer, the alteration of the interfacial properties of the electrodes upon peptide-antibody interaction was traced by faradaic electrochemical impedance spectroscopy (EIS) using a silicotungstic heteropolyacid, H(4)SiO(4).12WO(3), as a redox probe. The electrochemical behaviour of the redox probe was evaluated with cyclic voltammetry and EIS. The effect of milk protein or 4-mercaptophenol, which was used as post-blocking agents, on the performance of the immunosensor, was investigated. Treatment with 4-mercaptophenol resulted in immunoeffective electrodes that successfully tested in anti-serum samples. An optimum dilution ratio of the samples, where the effect of the matrix on the measuring signal is negligible, was also determined.

Bla g 6: a troponin C allergen from Blattella germanica with IgE binding calcium dependence.

BACKGROUND: The known cockroach allergens do not appear to account for the full repertoire of IgE responses. OBJECTIVE: To identify and investigate the importance of other Blattella germanica allergens contributing to cockroach allergy. METHODS: A B germanica cDNA library was screened with pooled sera from patients with cockroach allergy. Three isoallergens of troponin C (Bla g 6) were cloned and expressed in Pichia pastoris. Homology modeling was performed by using Swiss-Model. IgE responses to purified allergens were simultaneously measured in 104 sera by using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA. RESULTS: Three isoallergens, Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, were identified which share homology with insect troponin Cs and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium binding domains. Molecular models of Bla g 6 showed 2 structurally homologous lobes connected by a linker that confers flexibility to the allergen. The prevalence of IgE binding to recombinant Bla g 6 was 14%. Calcium depletion by 10 mmol/L ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid did not significantly affect IgE binding in most cases. Interestingly, addition of 10 mmol/L CaCl2 after calcium depletion increased IgE binding by approximately 2-fold, a finding not previously reported for calcium binding allergens. CONCLUSION: Bla g 6 is a troponin allergen with a calcium dependent IgE reactivity that may be involved in muscle contraction. CLINICAL IMPLICATIONS: Bla g 6 homologous allergens may occur among other insects and cause cosensitization or allergenic cross-reactivity.

Directed evolution for the development of conformation-specific affinity reagents using yeast display.

Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca(2+) binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca(2+)-bound (Ca(2+)-CaM) or Ca(2+)-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (K(d) = 0.8 nM) and specificity (>1000-fold) for Ca(2+)-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (K(d) = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform.

An interfering component in cardiac troponin I immunoassays-Its nature and inhibiting effect on the binding of antibodies against different epitopes.

OBJECTIVES: We recently reported on the occurrence of a common interfering factor (IF) that negatively affects determinations of cardiac troponin I (cTnI). The aim of the present investigation was to extend our initial finding by a detailed epitope-based determination of the location of IF and to reveal the approximate size and characteristics of IF. DESIGN AND METHODS: Two-site immunoassays using combinations of 16 monoclonal and 2 polyclonal cTnI antibodies and 1 monoclonal troponin C (TnC) antibody were used to measure the analytical recovery of cTnI or cTnI-TnC in serum samples. Gel filtration of serum samples containing IF was performed and the analytical recovery of cTnI in the fractions was determined. EDTA-plasma samples to which cTnI had been added and serum samples containing endogenous cTnI were also separated by gel filtration. RESULTS: The mean analytical recoveries of cTnI were 28.3% (range 7.5-55.1%) and of cTnI-TnC were 23.5% (range 8.7-51.8%) in samples containing IF when antibodies against midfragment epitopes of cTnI were used. The mean recovery of cTnI was 65.1% and 73.3% for antibodies with N- and C-terminal epitopes. Gel filtration of samples with low recovery of cTnI showed that the approximate molecular mass of IF was 50-200 kDa and that the cTnI elution profiles of samples containing IF were shifted towards higher molecular mass compared with samples with less IF. CONCLUSIONS: Antibodies against midfragment epitopes of cTnI are affected by IF to a considerable but variable extent, and the presence of IF can be demonstrated by gel filtration.

Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex.

Fourteen monoclonal antibodies (mAbs) against human cardiac troponin I (cTnI) were generated by commonly used experimental techniques. All these antibodies, as well as antibody 414 (HyTest), were specific for human cTnI. Fifteen antibodies thus obtained were tested in a sandwich cTnI immunofluorescence assay (altogether 196 combinations). Ten pairs giving the highest sensitivity were selected for further investigation. The effect of TnI-TnC complex formation on antibody interaction with antigen was analyzed. The formation of TnI-TnC complex results in a significant decrease of the interaction of mAbs with TnI for seven of 10 analyzed pairs of antibodies. Using two pairs of cTnI-specific mAbs, one that recognized only free cTnI but not cTnI complexed with cTnC, and another that could be used for measurement of total cTnI (free cTnI and cTnI in complex with cTnC), we demonstrated that the main part of cTnI in serum collected from acute myocardial infarction patients is presented in the complex from. We concluded that effective and reliable immunological detection of TnI is possible only when antibodies used for assay development recognize both free TnI and TnI complexed with other troponin components.