Species-specific Immunoassay Research Articles

Species-Specific Immunoassay Aids Identification of Pathogen and Tracks Infectivity in Foot Infection.

Conventional bacterial cultures frequently fail to identify the dominant pathogen in polymicrobial foot infections, in which Staphylococcus aureus is the most common infecting pathogen. Previous work has shown that species-specific immunoassays may be able to identify the main pathogen in musculoskeletal infections. We sought to investigate the clinical applicability of a S. aureus immunoassay to accurately identify the infecting pathogen and monitor its infectivity longitudinally in foot infection. We hypothesized that this species-specific immunoassay could aid in the diagnosis of S. aureus and track the therapeutic response in foot infections. From July 2015 to July 2019, 83 infected foot ulcer patients undergoing surgical intervention (debridement or amputation) were recruited and blood was drawn at 0, 4, 8, and 12 weeks. Whole blood was analyzed for S. aureus-specific serum antibodies (mix of historic and new antibodies) and plasmablasts were isolated and cultured to quantify titers of newly synthesized antibodies (NSAs). Anti-S. aureus antibody titers were compared with culture results to assess their concordance in identifying S. aureus as the pathogen. The NSA titer changes at follow-ups were compared with wound healing status to evaluate concordance between evolving host immune response and clinically resolving or relapsing infection. Analysis of serum for anti-S. aureus antibodies showed significantly increased titers of 3 different anti-S. aureus antibodies, IsdH (P = .037), ClfB (P = .025), and SCIN (P = .005), in S. aureus culture-positive patients compared with culture-negative patients. Comparative analysis of combining antigens for S. aureus infection diagnosis increased the concordance further. During follow-up, changes of NSA titers against a single or combination of S. aureus antigens significantly correlated with clinically resolving or recurring infection represented by wound healing status. In the management of foot infection, the use of S. aureus-specific immunoassay may aid in diagnosis of the dominant pathogen and monitoring of the host immune response against a specific pathogen in response to treatment. Importantly, this immunoassay could detect recurrent foot infection, which may guide a surgeon's decision to intervene. Level II, prospective comparative study.

Species-Specific Immunoassay for Diagnosis and Tracking Treatment Response in Foot and Ankle Infection

Category:Basic Sciences/Biologics; DiabetesIntroduction/Purpose:Staphylococcus aureus is the major pathogen foot and ankle infections and osteomyelitis (>50%). There are currently no sensitive and specific diagnostic tools for monitoring a pathogen’s ongoing infection or providing prognostic measures. We have developed a novel immunoassay for S. aureus, and have applied this to diagnose and monitor its infectivity. We hypothesize that: 1) the species-specific immunoassay can serve as a reliable diagnostic tool for S. aureus foot and ankle infections and 2) the immunoassay provides a measure of treatment response and prognosis of clinical outcome to antibiotics therapy for S. aureus foot and ankle infections.Methods:From July 2015 to July 2019, 83 infected diabetic foot ulcer (DFU) patients undertaking surgical treatment were recruited. Blood were drawn from subjects at initial, 4-week, 8-week, and 12-week after surgery. Clinical wound healing status was determined by a fellowship-trained orthopaedic foot and ankle surgeon. Serum antibodies and plasmablast cultured antibodies (newly synthesized antibodies: NSA) were harvested. Eight unique S. aureusantigens from distinct functional classes were used for the immunoassay. All serum and NSA samples were run on a flow cytometer (Bio-Plex 200; Bio-Rad, Life Sciences Research) in duplicates and assessed for predictive ability in discriminating infection status and healing status using receiver operating characteristic (ROC) curve analysis, with accuracy summarized by the area under the ROC curve (AUC). Nonparametric estimates and 95% confidence intervals for the AUC were computed for each predictor along with p-values for testing the significance of each AUC.Results:Analysis of serum immunoassay showed significant difference in three anti-S. aureus antigens titers (IsdH (p = 0.037; AUC = 0.638), ClfB (p = 0.025; AUC = 0.644), and SCIN (p = 0.005; AUC = 0.677)) between S. aureus infected versus non- S. aureus infected DFU patients at initial presentation. NSA immunoassay showed elevation of two different S. aureus specific antigens, IsdH and LukS-PV, for S. aureus infected versus non- S. aureus infected DFU patients in ratios of approximately five and four, respectively. Changes of NSA based anti-S. aureus antibody titers over 12 weeks period, as single antigen or in combination, significantly correlated with clinical resolution of infection and wound healing status. Four anti-S. aureus antigen combinations achieved the highest AUC (Figure 1).Conclusion:Our results demonstrate that both the serum and NSA immunoassay can diagnose S. aureus infected DFUs. Furthermore, changes in NSA titers over a period of time against various S. aureus specific antigens significantly correlated with clinical representation of infection and wound healing status. The novel species-specific immunoassay can serve as a promising diagnostic system, tracking tool and prognostic potential in management of S. aureus associated foot and ankle infection.

Evaluation of yolk protein levels as estrogenic biomarker in bivalves; comparison of the alkali-labile phosphate method (ALP) and a species-specific immunoassay (ELISA)

Altered concentration of the vertebrate yolk protein precursor vitellogenin is a recognized biomarker for endocrine disruption in fish, and within recent years yolk protein alteration has also been associated with endocrine disruption in bivalves. Species-specific, direct and sensitive methods for quantification of vitellogenin in fish have been available for years whereas bivalve yolk protein levels have been estimated indirectly by alkali-labile phosphate (ALP) liberated from high molecular weight proteins because the sequence and biochemical structure of most bivalve yolk proteins are unknown. By applying a species-specific enzyme-linked immunosorbent assay (ELISA) for accurate determination of yolk protein level the impact of 17β-estradiol (57, 164 and 512ng/L) on the freshwater bivalve Unio tumidus was investigated and compared with ALP estimations. Seven weeks of exposure during the pre-spawning and spawning period had no consistent effect on yolk protein concentration in hemolymph, and ALP levels in hemolymph also remained unchanged in both males and females. Further, basal male and female ALP levels were indistinguishable whereas the ELISA demonstrated that yolk protein levels of females exceeded male levels at the time of sampling, although male basal levels were high compared to fish. Altogether it is shown that individual ALP levels do not reflect yolk protein levels and hence hemolymph ALP levels cannot serve as biomarker for estrogenic exposure during the pre-spawning and spawning period in U. tumidus. The necessity of sensitive and validated biomarkers for reliable interpretation of data and the utility of ALP and yolk protein levels as biomarkers in bivalves are discussed.

Universal Immunogenicity Validation and Assessment During Early Biotherapeutic Development to Support A Green Laboratory

Immunogenicity support during nonclinical biotherapeutic development can be resource intensive if supported by conventional methodologies. A universal indirect species-specific immunoassay can eliminate the need for biotherapeutic-specific anti-drug antibody immunoassays without compromising quality. By implementing the R's of sustainability (reduce, reuse, rethink), conservation of resources and greener laboratory practices were achieved in this study. Statistical analysis across four biotherapeutics supported identification of consistent product performance standards (cut points, sensitivity and reference limits) and a streamlined universal anti-drug antibody immunoassay method implementation strategy. We propose an efficient, fit-for-purpose, scientifically and statistically supported nonclinical immunogenicity assessment strategy. Utilization of a universal method and streamlined validation, while retaining comparability to conventional immunoassays and meeting the industry recommended standards, provides environmental credits in the scientific laboratory. Collectively, individual reductions in critical material consumption, energy usage, waste and non-environment friendly consumables, such as plastic and paper, support a greener laboratory environment.

Antifouling Surface Layers for Improved Signal-to-Noise of Particle-Based Immunoassays

A 10-fold improvement in the signal-to-noise (S/N) ratio of an optically encoded silica particle-based immunoassay was achieved through incorporating a protein resistant poly(ethylene glycol) (PEG) surface layer and optimizing antibody immobilization conditions. PEG was activated using 2,2,2-trifluoroethanesulfonyl chloride (tresyl) and required a minimum reaction time of 1.5 h. The activated PEG had a reactive half-life of approximately 5 h when stored in acidified dimethyl sulfoxide (DMSO). By increasing the protein incubation time and concentration, a maximum antibody loading on the particle surface of 1.6 x 10(-2) molecules per nm(2) was achieved. The assay S/N ratio was assessed using a multiplexed multicomponent optically encoded species-specific immunoassay. Encoded particles were covalently grafted or nonspecifically coated with either bovine or mouse IgG for the simultaneous detection of complementary anti-IgG "target" or uncomplementary anti-IgG "noise". The versatility and potential as a serum-based assay platform was demonstrated by immobilizing either a polyclonal antibody or an engineered single-chain variable fragment (scFv) capture probe on particles for the detection of the ovarian cancer biomarker, mesothelin (MSLN). The MLSN antigen was spiked into PBS buffer or 50% human serum. Both capture probe orientations, and media conditions showed similar low level detection limits of 5 ng/mL; however, a 40% decrease in maximum signal intensity was observed for assays run in 50% serum.

Rapid method for the detection of storage mites in cereals: feasibility of an ELISA based approach

This paper describes the development of rapid immunodiagnostic tests for the detection of storage mite infestations in cereals and cereal products. The study's first phase (proof of concept) involved the production of a species-specific enzyme-linked immunoassay (ELISA) for the flour mite, Acarus siro (L.), a major pest of stored commodities. The specificity of this new assay was assessed against key stored product contaminants (13 species of mites of which three were predatory, five species of insects and five species of fungi) in the presence and absence of grain. The assay was species-specific (no cross-reactivity to other storage contaminants) and was unaffected by the presence of cereal antigens in the extract. In the study's second phase, species- and genera-specific ELISAs were developed for a range of key storage mite pests: the cosmopolitan food mite (Lepidoglyphus destructor), the grocers' itch mite (Glycyphagus domesticus), the grainstack mite (Tyrophagus longior), mites of the Tyrophagus and Glycyphagus generas, and all storage mites. All tests were demonstrably specific to target species or genera, with no cross-reactions observed to other storage pest contaminants or cereals. The final, validation phase, involved a comparative assessment of the species-specific A. siro and the genus-specific Tyrophagus ELISAs with the flotation technique using laboratory and field samples. Both ELISAs were quantitative (0-30 mites per 10 g wheat) and produced good comparative data with the flotation technique (A. siro r(2)=0.91, Tyrophagus spp. r(2)=0.99).

Monoclonal antibodies specific for hippurate hydrolase of Campylobacter jejuni.

Eleven monoclonal antibodies raised against recombinant Campylobacter jejuni hippurate hydrolase were tested for binding to lysates from 19 C. jejuni strains, 12 other Campylobacter strains, and 21 non-Campylobacter strains. Several monoclonal antibodies bound to C. jejuni but not to other Campylobacter species and may be useful in a species-specific immunoassay.

Detection of antibodies toChlamydia trachomatis with peptide-based species-specific enzyme immunoassay

Objective: We have evaluated the sensitivity and specificity of a new synthetic peptide-based species-specific enzyme immunoassay (EIA) for detection of Chlamydia trachomatis IgG and IgA antibodies.