Clinical Immunodiagnostics Research Articles

Toxoplasma gondii induces robust humoral immune response against cyst wall antigens in chronically infected animals and humans

Toxoplasma gondii differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission. Strong humoral immune response has been reported against tachyzoite antigens, however, antibody-mediated response towards bradyzoite antigens is poorly characterized. This work aimed to study the humoral immune response towards bradyzoite and associated cyst wall antigens particularly CST1. The immunoreactivity of 404 goats, 88 sheep and 92 human sera to recombinant (CST1 and SRS9) and native proteins of encysted bradyzoite along with well-established tachyzoite antigens (SAG1 and GRA7) was determined using ELISA, Western blot and immunofluorescence analysis (IFA). ELISA results revealed nearly 50% of sera contain T. gondii specific antibodies. Results were further validated using Western blot and IFA. T. gondii positive sera predominantly recognized the cyst wall besides the known tachyzoite surface antigens. The presence of CST1 antibodies in seropositive samples were in line with the staining patterns which were consistent with CST localization. Notably, T. gondii IgM- IgG+ sera recognize the cyst wall whereas IgM + IgG-sera recognize tachyzoite antigens indicating acute infection consistent with presence of parasite DNA. The study demonstrates a strong humoral response against bradyzoite associated cyst wall antigens across naturally infected animals and humans. CST1 emerged as a key immunomodulatory antigen which may have direct implications for clinical immunodiagnostics.

Immunodiagnostics: Major Advances and Future Insights

There have been tremendous advances in immunodiagnostics during the last five decades, which have led to the emergence of potential diagnostic technologies, new immunoassay formats and diagnostic platforms. The radioimmunoassay and Enzyme Linked Immunosorbent Assay (ELISA) were the predominant techniques during the initial era of immunodiagnostics. The strenuous research efforts during the last three decades have enabled the development of potential antibodies for a wide range of analytes. Till date,t ELISA has been the gold standard of clinical immunodiagnostics, which has also been used very frequently for industrial analysis, environmental monitoring, food analysis and bioanalytical sciences. Despite the numerous advances in the field, it is difficult for any other technique to achieve the same high sensitivity, specificity, precision and throughput as ELISA. Various robotic work stations have been devised to perform automated ELISAs in high volume settings, such as in hospitals and industry. Similarly, the Microtiter Plate (MTP) readers have been improved significantly in terms of optics, technology and data analysis. In addition to conventional 96-well MTP format, various high throughput formats, such as 384 and 1536 wells, have also been devised for specific needs. The last two decades have seen considerable improvements in antibody immobilization strategies [1-3], which have significantly improved the analytical performance of ELISA in terms of higher sensitivity, greater detection range, lower limit of detection and reduced assay duration [4-6]. Similarly, the sample preparation strategies and screening of immunological components have also been critically improved, which enables highly reproducible immunoassays [7,8]. The development of highly sensitive enzyme substrates have led to ultrasensitive ELISAs and chemiluminescent immunoassays [9], while the use of superior fluorescent dyes improved the fluorescent immunoassays. Additionally, the use of infra-red dyes, quantum dots, gold nanoparticles, beads, and other nanomaterial/nanocomposites enabled the development of new ELISA formats, such as the naked-eye ELISA that was demonstrated using gold nanoparticles [10].

Prospects of Nanotechnology in Clinical Immunodiagnostics

Nanostructured materials are promising compounds that offer new opportunities as sensing platforms for the detection of biomolecules. Having micrometer-scale length and nanometer-scale diameters, nanomaterials can be manipulated with current nanofabrication methods, as well as self-assembly techniques, to fabricate nanoscale bio-sensing devices. Nanostructured materials possess extraordinary physical, mechanical, electrical, thermal and multifunctional properties. Such unique properties advocate their use as biomimetic membranes to immobilize and modify biomolecules on the surface of nanoparticles. Alignment, uniform dispersion, selective growth and diameter control are general parameters which play critical roles in the successful integration of nanostructures for the fabrication of bioelectronic sensing devices. In this review, we focus on different types and aspects of nanomaterials, including their synthesis, properties, conjugation with biomolecules and their application in the construction of immunosensing devices. Some key results from each cited article are summarized by relating the concept and mechanism behind each sensor, experimental conditions and the behavior of the sensor under different conditions, etc. The variety of nanomaterial-based bioelectronic devices exhibiting novel functions proves the unique properties of nanomaterials in such sensing devices, which will surely continue to expand in the future. Such nanomaterial based devices are expected to have a major impact in clinical immunodiagnostics, environmental monitoring, security surveillance and for ensuring food safety.

Fluorescence Single-Molecule Counting Assays for High-Sensitivity Detection of Cytokines and Chemokines

A recent focus in clinical immunodiagnostics is to improve sensitivity of assays for rare circulating protein biomarkers. The ability to detect low concentrations of certain biomarkers translates to diagnosis of disease at an earlier stage, which can positively impact prognosis and disease management (1)(2)(3). In research (for example in the discovery and development of new biomarkers), targeted assays for individual proteins are often required before multiplexing strategies are developed. Cross-reactivity of antibodies often compromises assay sensitivity in multiplexed immunoassays(4). Ultrasensitive individual assays consume only small sample volumes, a characteristic that can be advantageous in the case of scarce archival clinical samples. The development of single-molecule detection approaches has provided new opportunities for improving immunoassay sensitivity and miniaturization (5). Fluorescence confocal microscopy with laser-induced excitation enables single-molecule detection in extremely small interrogation zones (on the order of femtoliters). By reducing interrogation zones to these dimensions and carefully tuning the timescales of the data acquisition and molecular flow rates, background interference is decreased and individual molecules are readily recorded as discrete fluorescence bursts above background(6). Counting these bursts, or molecular events, by applying fluorescence thresholds on the basis of the internally derived background noise generates experimental data whose precision is dictated by counting statistics. Hence, sample read times can be adjusted to achieve a desired counting precision. The combination of small interrogation volumes and short sample read times can also lead to rapid analysis. Despite the advantages of single-molecule detection and the development of single-molecule detection instruments, adaptation of this technology to quantification of serum or plasma analytes has only scarcely been reported in the literature [for example, (7)]. Building on our previous work(8), we constructed single-molecule immunoassays (SMIAs) that use immune capture of analytes on microparticles, tagging with biotinylated antibodies, and quantification of fluorophore-labeled …

Luminescent lanthanide chelates—a way to more sensitive diagnostic methods

The unique luminescence properties of certain lanthanides are valuable features in aiming at more sensitive label technologies for clinical diagnostic use. The use of these properties in bioanalytical assays requires development of specific chelate technologies for labelling, i.e. binding of the ion to bioaffinity reagents, carrying it through the assay procedure, creating the optimal environment for emission and so forth. So far the most successful lanthanide label technology is composed of sequential use of two different chelate systems: a bifunctional polyaminopolycarboxylate derivative used for labelling of bioaffinity reactants with the lanthanide ion and a β-diketone-TOPO chelate optimized for time-resolved fluorometric determinations. This technology has achieved an established position as a highly sensitive method in in vitro clinical immunodiagnostics. Subsequently, a number of alternative approaches have been developed, including stable and highly luminescent chelates, which can be directly coupled to bioaffinity reactants to form luminescent tracers. These chelates are invaluable in studies aiming at sensitive and quantifiable localizations of analytes directly in situ, e.g. on tissue sections or cell surfaces.

Monoclonal antibodies in clinical immunology.

Advances in clinical immunodiagnostics resulting from the application of hybridoma technology are starting to appear. Monoclonal antibodies are beginning to displace their antiquated progenitors, polyclonal antisera, in many facets of immunology. Their homogeneity, specificity, and availability make hybridoma-derived antibodies the immunological reagents of the future in immunoassays, immuno-affinity chromatography, immunotherapy, and areas yet to be defined.