Mite-specific AIT should rely upon a mixture of D. pteronyssinus and D. farinae extracts, manufactured from both feces and bodies. Such a combination is appropriate to treat children and adult Dermatophagoides-allergic patients from Asia, Europe, and North America.
We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria-Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of E. coli spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable E. coli in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection.
According to the World Health Organization, food safety is an essential public health priority. In this context, we report a relevant proof of feasibility for the indirect specific detection of bacteria in food samples using unlabeled phage amplification coupled to ESI mass spectrometry analysis and illustrated with the model phage systems T4 and SPP1. High-resolving power mass spectrometry analysis (including bottom-up and top-down protein analysis) was used for the discovery of specific markers of phage infection. Structural components of the viral particle and nonstructural proteins encoded by the phage genome were identified. Then, targeted detection of these markers was performed on a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. E. coli at 1 × 10(5), 5 × 10(5), and 1 × 10(6) CFU/mL concentrations was successfully detected after only a 2 h infection time by monitoring phage T4 structural markers in Luria-Bertani broth, orange juice, and French bean stew ("cassoulet") matrices. Reproducible detection of nonstructural markers was also demonstrated, particularly when a high titer of input phages was required to achieve successful amplification. This strategy provides a highly time-effective and sensitive assay for bacterial detection.
BackgroundHouse dust mites (HDMs) such as Dermatophagoides farinae and D. pteronyssinus represent major causes of perennial allergy. HDM proteomes are currently poorly characterized, with information mostly restricted to allergens. As of today, 33 distinct allergen groups have been identified for these 2 mite species, with groups 1 and 2 established as major allergens. Given the multiplicity of IgE-reactive mite proteins, potential additional allergens have likely been overlooked.ObjectiveTo perform a comprehensive characterization of the transcriptomes, proteomes and allergomes of D. farinae and D. pteronyssinus in order to identify novel allergens.MethodsTranscriptomes were analyzed by RNA sequencing and de novo assembly. Comprehensive mass spectrometry-based analyses proteomes were combined with two-dimensional IgE reactivity profiling.ResultsTranscripts from D. farinae and D. pteronyssinus were assembled, translated into protein sequences and used to populate derived sequence databases in order to inform immunoproteomic analyses. A total of 527 and 157 proteins were identified by bottom-up MS analyses in aqueous extracts from purified HDM bodies and fecal pellets, respectively. Based on high sequence similarities (>71% identity), we also identified 2 partial and 11 complete putative sequences of currently undisclosed D. pteronyssinus counterparts of D. farinae registered allergens. Immunoprofiling on 2D-gels revealed the presence of unknown 23 kDa IgE reactive proteins in both species. Following expression of non-glycosylated recombinant forms of these molecules, we confirm that these new allergens react with serum IgEs from 42% (8/19) of HDM-allergic individuals.ConclusionsUsing combined transcriptome and immunoproteome approaches, we provide a comprehensive characterization of D. farinae and D. pteronyssinus allergomes. We expanded the known allergen repertoire for D. pteronyssinus and identified two novel HDM allergens, now officially referred by the International Union of Immunological Societies (IUIS) Nomenclature Subcommittee as Der f 36 and Der p 36.
Multispecific antibodies,
which target multiple antigens at once,
are emerging as promising therapeutic entities to offer more effective
treatment than conventional monoclonal antibodies (mAbs). However,
these highly complex mAb formats pose significant analytical challenges.
We report here on the characterization of a trispecific antibody (tsAb),
which presents two isomeric forms clearly separated and identified
with size exclusion chromatography coupled to native mass spectrometry
(SEC-nMS). Previous studies showed that these isomers might originate
from a proline
cis
/
trans
isomerization
in one Fab subunit of the tsAb. We combined several innovative ion
mobility (IM)-based approaches to confirm the isomeric nature of the
two species and to gain new insights into the conformational landscape
of both isomers. Preliminary SEC-nIM-MS measurements performed on
a low IM resolution instrument provided the first hints of the coexistence
of different conformers, while complementary collision-induced unfolding
(CIU) experiments evidenced distinct gas-phase unfolding behaviors
upon activation for the two isomers. As subtle conformational differences
remained poorly resolved on our early generation IM platform, we performed
high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the
coexistence of two conformers. The
cis
/
trans
equilibrium was further tackled by exploiting the IM
n
slicing capabilities of the cIM-MS instrument. Altogether, our results
clearly illustrate the benefits of combining state-of-the-art nMS
and IM-MS approaches to address challenging issues encountered in
biopharma. As engineered antibody constructs become increasingly sophisticated,
CIU and cIM-MS methodologies undoubtedly have the potential to integrate
the drug development analytical toolbox to achieve in-depth conformational
characterization of these products.
Proteomics encompasses a variety of approaches unraveling both the structural features, post-translational modifications, and abundance of proteins. As of today, proteomic studies have shed light on the primary structure of about 850 allergens, enabling the design of microarrays for improved molecular diagnosis. Proteomic methods including mass spectrometry allow as well to investigate protein-protein interactions, thus yielding precise information on critical epitopes on the surface of allergens. Mass spectrometry is now being applied to the unambiguous identification, characterization, and comprehensive quantification of allergens in a variety of matrices, as diverse as food samples and allergen immunotherapy drug products. As such, it represents a method of choice for quality testing of allergen immunotherapy products.
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