Solid-phase Immunoassay Research Articles

Solid-phase and bead-based cytokine immunoassay: A comparison

Cytokines and chemoattractive cytokines (chemokines) are present in a wide variety of body fluids such as plasma, cerebrospinal fluid, bronchoaveolar fluid, amniotic fluid, synovial fluid, middle ear effusion fluid, and urine. Cytokines can be detected using classical solid-phase sandwich immunoassays such as enzyme-linked immunosorbent assay (ELISA) or with a bead based multiplex immunoassay (MIA). The physical chemical properties of the different body fluids (such as pH and total protein content) differ, which may have an impact on the outcome of the cytokine assay. Both ELISA as well as MIA cytokine detection systems are constructed by sandwiching the protein of interest between a capture and reporter antibody. When the biological sample contains heterophilic antibodies (such as in patients with auto-immune diseases), these non-specific antibodies can cause false positive results. During pathological conditions, cytokines may be found over a wide concentration range; likewise have to cover this dynamic range in a similar fashion. The correct (statistical) analysis of standard curves and (multiplexed) data are critical for proper interpretation. Classical ELISA based cytokine assays are robust, easy to use and very well suited for measurement of single cytokines. Due to an increased interest in the integral approach to understand biological processes (the omics era), multiplex immunoassays for detection of cytokines and the interpretation of these assays are gaining popularity.

Flow injection chemiluminescence immunoassay for 17β-estradiol using an immunoaffinity column

A new flow injection chemiluminescent immunoassay was developed for the detection of 17beta-estradiol (E2). The method uses p-iodophenol (PIP) as enhancer and is based on a solid-phase immunoassay format in which an E(2)-OVA immobilized immunoaffinity column inserted in the flow system is used to trap unbound horseradish peroxidase (HRP)-labeled anti-E2 antibody after an off-line incubation of E2 with HRP-labeled anti-E2 antibody. The trapped enzyme conjugate was detected by injecting substrates to produce an enhanced chemiluminescence (CL) response. The linear range for E2 was 10.0-1,000.0 ng mL(-1) with a correlation coefficient of 0.996 and a detection limit of 3.0 ng mL(-1). The sampling and chemiluminescence detection time for one sample was 400 s after a pre-incubation procedure of 30 min. Serum samples detected by this method were in good agreement with the results obtained by EIA with E2-biotin.

Detection of Antinuclear Antibodies by Solid-Phase Immunoassays and Immunofluorescence Analysis

Detection of Antinuclear Antibodies by Solid-Phase Immunoassays and Immunofluorescence Analysis

Gold-Coated Magnetic Particles for Solid-Phase Immunoassays: Enhancing Immobilized Antibody Binding Efficiency and Analytical Performance

The preparation and characterization of gold-coated magnetic particles are described for use as more efficient solid-phase materials in immunoassay development. A thin gold coating on commercial tosylated magnetic polystyrene particles (4.5 microm) is achieved via an electroless plating method involving initial reaction of the particles with Sn(II), followed by redox deposition of Ag0, that serves as a catalytic site for the subsequent reduction of Na3Au(SO3)2 in the presence of formaldehyde to yield the adhered gold layer. Scanning electron microscopy, energy-dispersive X-ray analysis, and X-ray photoelectron spectroscopy indicate the presence of the desired Au0 outer layer. To characterize the improved yield of antibody binding sites on such gold-coated phases, the modified particles are reacted with the free thiols of Fab' fragments of an anti-alkaline phosphatase (ALP) antibody to orient all the antigenic binding sites in a favorable direction. After equilibration with ALP, the amount of ALP bound to the surface of such particles is nearly 2.5-fold greater than on non-gold-coated particles possessing the same amount of immobilized anti-ALP Fab', but oriented randomly on the surface. The new gold-coated magnetic particles are further used as a solid phase for developing a sandwich-type enzyme immunoassay to detect C-reactive protein (CRP) using horseradish peroxidase as the enzyme label. The gold-coated magnetic particles with anti-CRP monoclonal Fab' reagents provide assays with enhanced assay slope (1.8-fold), lower nonspecific adsorption, and a detection limit improvement of nearly 10-fold (0.14 vs 1.9 ng/mL) compared to the same Fab' anti-CRP immobilized on the initial tosylated polystyrene magnetic particles. The improved assay performance is attributed to the more favorable binding orientation of the self-assembled monolayer of Fab' fragments on the gold-coated particles compared to the random orientation on the non-gold-coated surfaces.

Electrochemical Microbead-Based Immunoassay Using an (η5-Cyclopentadienyl)tricarbonylmanganese Redox Marker Bound to Bovine Serum Albumin

A first example of the solid-phase immunoassay of a high-weight antigen bovine serum albumin (BSA) using an (eta(5)-cyclopentadienyl)tricarbonylmanganese (cymantrene) redox probe is presented. The electrochemical detection is based on the impedance measurements of a one-electron reversible reduction of the organometallic probe. The microbead-based immunoassay is discussed for two types of microbeads with different diameters (2.5 and 90 microm) and capabilities to bind the immunoglobulins (2.4 and 10 microg/mg of beads). The use of larger agarose microbeads allows the formation of an antigen-antibody complex at the surface of microbeads directly dispersed in the analyzed solution. No additional separation step is necessary for the electrochemical competitive immunoassay analysis of BSA. The presence of agarose beads in the analyzed solution has no effect on the electrochemical signal from labeled BSA released from the antigen-antibody complex.

Chimeric synthetic peptides as antigens for detection of antibodies to Trypanosoma cruzi

Six chimeric synthetic peptides (QCha-1, QCha-2, QCha-3, QCha-4, QCha-5, and QCha-6) incorporating antigenic sequences of two immunodominant repeat B-cell epitopes of Trypanosoma cruzi were synthesized by conventional solid-phase peptide synthesis. The antigenic activity of these peptides was evaluated by UltramicroEnzyme-linked immunosorbent assay (UMELISA) by using panels of positive Chagasic sera ( n = 82), while specificity was evaluated with samples from healthy blood donors ( n = 44) and patients with other infectious diseases ( n = 86). The antigenicity of the chimeric peptides in solid-phase immunoassays was compared with that of the monomeric peptides. Data demonstrated that the chimeric peptide QCha-5 was the most reactive because it detected antibodies to parasite efficiently. The results indicate that chimeric peptide as coating antigen is very useful for the immunodiagnosis of Chagas’ disease.

A solid-phase immunoassay of protease-resistant prion protein with filtration blotting involving sodium dodecyl sulfate

The precise diagnosis for bovine spongiform encephalopathy (BSE) is crucial for preventing new transmission to humans. Several testing procedures are reported for determining protease-resistant prion protein in various tissues as a major hallmark of prion diseases such as BSE, scrapie, and Creutzfeldt–Jakob disease. However, contamination of materials from tissues or degradation of the specimens sometimes disturbs the accuracy of the assay. Here, we have developed a novel method for solid-phase immunoassay of the disease-specific conformational isoform, PrP Sc, using filtration blotting of protein in the presence of sodium dodecyl sulfate (SDS) followed by a filtration-based immunoassay with a single anti-prion protein antibody, together with the improved fractionation procedure involving high concentrations of surfactant/detergent. The SDS/heat treatment renders unfolded PrP Sc quantitative retention on a polyvinylidene difluoride filter and allows enhancement of the analyte signal with immunodetection; thus, all of the tested specimens are determined with 100% accuracy. In addition, the immunoassay is completed in approximately 1 h, indicating its usefulness not only for the screening of BSE specimens but probably also for the postmortem BSE diagnosis of fallen stock as the antibody recognizes the core part of PrP Sc. The solid-phase immunoassay method, including the filtration blotting with SDS, would be applicable to determining even more sensitively proteins other than PrP Sc, especially those having rigid conformations.

Monoclonal Antibodies to Ricin:In VitroInhibition of Toxicity and Utility as Diagnostic Reagents

Monoclonal antibodies (MAbs) against ricin toxin (RT) and its subunits were produced in mice. The MAbs were initially selected based upon the ability to either bind ricin or the individual subunits in a solid-phase enzyme-linked immunosorbent assay (ELISA). Several candidates were selected for further evaluation, including their ability to inhibit ricin intoxication in vitro and their utility as immunodiagnostic reagents. Although their ability to capture antigen when bound to the solid phase was poor, some MAbs demonstrated potential utility as detection reagents in solid-phase immunoassays. Several MAbs were also able to inhibit ricin-mediated eukaryotic cell cytotoxicity in vitro. These MAbs may prove useful for preventing and/or treating ricin intoxication.

Innovative, Multifunctional Sequential Oligopeptide Carriers Socn-I and SOCn-II: Functions-Technology-Perspectives

An innovative type of multifunctional helicoid artificial carriers, formed by the repetitive Lys-Aib-Gly (SOC(n)-I) or by the Aib-Lys-Aib-Gly (SOC(n)-II), with structural rigidity and regularity were successfully applied in our laboratory for anchoring antigenic/immunogenic peptides. The carriers, designed to display a predetermined 3D structure, adopt the 3(10) helical conformation, while the attached peptides preserve their original "active" conformation. The constructed conjugates were used as substrates in solid phase immunoassays, as well as for generating potent and specific immune responses.

Increased Prevalence of Latex-Sensitization Among Children With Chronic Renal Failure

Dehlink E, Prandstetter C, Eiwegger T, Putschogl B, Urbanek R, Szepfalusi Z. Allergy. 2004;59:734–738To assess the prevalence of latex sensitization and identify risk factors for latex sensitization among children with chronic renal failure.Ninety-three patients (44 boys and 49 girls; median age: 10.5 ± 6.0 years) with chronic renal failure who presented to the University of Vienna Children’s Hospital Nephrology Clinic between 1997 and 2000.Latex sensitization was assessed by a questionnaire-based history and measurement of total and latex-specific serum IgE by solid-phase immunoassay. Patients and parents were queried regarding the etiology of renal failure, age at onset, number of renal transplantations, surgical procedures, hemodialysis, clinical symptoms with latex exposure, personal and family history of allergy, and history of pacifier use. Patient responses were compared with medical histories and were consistent. Patients were designated as latex sensitized if their latex-specific serum IgE was ≥0.35 kilounits of allergen-specific IgE per liter (kUA/L). Neither skin-prick testing nor provocation testing was performed.Of the 93 patients enrolled, 10 (10.8%) were found to have latex-specific IgE levels (0.35–9.44 kUA/L.) Of those, only 1 patient reported clinical symptoms on latex exposure compared with 4 patients with no demonstrable latex-specific IgE. No reactions to latex were reported to occur during medical care. A personal or family history of allergy, a greater number of urogenital surgeries, and hemodialysis were reported more frequently in latex-sensitized children. Gender, age at enrollment, age at first urogenital surgery, renal transplantation, and the use of pacifiers did not differ between latex-sensitized and -nonsensitized children.The prevalence of latex sensitization among children with chronic renal failure is greater than that previously reported for the general pediatric population (10 of 93 vs 8 of 1175 in an unselected pediatric population). Eight of the 10 sensitized patients in this study had renal disease diagnosed within the first year of life and therefore had early and repeated exposures to latex. The small sample size prevented detection of significant associations with any determinant of renal disease or definite risk factor for sensitization.Atopy and repeated exposure to latex allergen have been previously associated with sensitization to latex. Children with chronic renal failure are frequently exposed to latex and therefore are likely at increased risk. A larger study population would be required to further characterize this increased risk. This study raises interesting questions regarding the impact of both timing and cumulative amount of latex exposure for all children with chronic illnesses.

Effects of exercise training on insulin-like growth factor-I expression in the skeletal muscle of non-cachectic patients with chronic heart failure

Chronic heart failure (CHF) is associated with progressive muscle atrophy and reduced local expression of insulin-like growth factor I (IGF-I). The present study was designed to test the hypothesis that the local deficiency of IGF-I in the skeletal muscle of patients with CHF would respond to a 6-months aerobic training intervention. Therefore, 18 patients [mean age 52.4 (SD 4.8) years, left ventricular ejection function (LVEF) 27 (SD 6)%] were prospectively randomized to either 6 months of training or sedentary lifestyle. Serum levels of growth hormone (GH) were measured by immunofluorometric assay, IGF-I by competitive solid phase immunoassay. IGF-I expression was assessed in vastus lateralis biopsies by real-time PCR. Exercise training led to a significant increase in peak oxygen uptake by 26% [from 20.3 (SD 3.3) ml/kg per min to 25.5 (SD 5.7) ml/kg per min, P=0.003 versus control]. Local expression of IGF-I increased significantly after exercise training by 81% [from 6.3 (SE 0.8) to 11.4 (SE 1.4) relative units, P=0.007 versus control] while IGF-I receptor expression was reduced by 33% [from 20.0 (SE 2.1) to 13.8 (SE 1.7) relative units, P=0.008 versus control]. Serum growth hormone (GH) rose modestly from 0.12 (SE 0.07) to 0.65 (SE 0.37) ng/ml in the training group (P=0.043 versus baseline), however, this change was not significant compared to the control group (P=0.848). IGF-I serum levels remained virtually unchanged. Exercise training improves local IGF-I expression without significant changes of systemic parameters of the GH/IGF-I axis. These findings indicate that exercise training has the therapeutic potential to attenuate peripheral skeletal muscle alterations in particular with respect to local IGF-I expression in patients with moderate CHF.

Protein microarrays for the diagnosis of allergic diseases: State-of-the-art and future development Protein-Biochips für die Diagnose allergischer Erkrankungen – Stand der Technik und zukünftige Entwicklungen

Abstract In the emerging field of Functional Proteomics, protein microarrays are considered to be one of the most promising tools for the simultaneous analysis of the a) abundance, b) function, and c) interaction of proteins on a system-wide scale. Resting on the technological grounds of widely used DNA biochips, the great power of microarray-based miniature solid-phase immunoassays lies in their potential to investigate in parallel large numbers of analyte pairs in a variety of biological samples. Consequently, this has fuelled aspirations that protein micro-arrays may serve as tools for the high-throughput functional investigation of complete proteomes and, moreover, that they will develop into promising candidates for innovative in-vitro diagnostic (IVD) applications. To date, published examples of protein microarrays for IVD purposes have included tests for allergy, autoimmune and infectious diseases. Here, we discuss recent advancements in the development of protein microarrays for the profiling of IgE antibodies in the diagnosis of Type 1-related allergic diseases. Im ständig wachsenden Forschungsgebiet der funktionellen Proteomik zählen Protein-Biochips aus einer Reihe innovativer Methoden für die quantitative und funktionelle Bestimmung von Proteininteraktionen auf systemischer Ebene heute zu den aussichtsreichsten Kandidaten. Miniaturisierte Festphasen-Immunoassays, die analog zur etablierten DNA-Chip-Technologie entwickelt wurden, entfalten ihr Potential in der hoch parallelen Untersuchung unterschiedlichster Liganden-Paare in biologischem Ausgangsmaterial. Es besteht die berechtigte Hoffnung, dass Protein-Biochips in Zukunft durch den hohen Probendurchsatz und die enorme Zahl der generierten Daten den Bereich der Proteomik revolutionieren werden. Des Weiteren sind sie viel versprechende Kandidaten für neue und innovative in-vitro-Diagnostika (IVD). In mehreren aktuellen Publikationen wurde veranschaulicht, dass Protein-Biochips für die Diagnose von allergischen, autoimmunen oder infektiösen Erkrankungen wertvolle Beiträge leisten können. In diesem Artikel beleuchten wir neue Ansätze und Entwicklungen im Bereich der Diagnostik von IgE-vermittelten Typ 1-Allergien mit Hilfe von Allergen-Biochips.

Antibodies from Inflamed Central Nervous System Tissue Recognize Myelin Oligodendrocyte Glycoprotein

Autoantibodies to myelin oligodendrocyte glycoprotein (MOG) can induce demyelination and oligodendrocyte loss in models of multiple sclerosis (MS). Whether anti-MOG Abs play a similar role in patients with MS or inflammatory CNS diseases by epitope spreading is unclear. We have therefore examined whether autoantibodies that bind properly folded MOG protein are present in the CNS parenchyma of MS patients. IgG was purified from CNS tissue of 14 postmortem cases of MS and 8 control cases, including cases of encephalitis. Binding was assessed using two independent assays, a fluorescence-based solid-phase assay and a solution-phase RIA. MOG autoantibodies were identified in IgG purified from CNS tissue by solid-phase immunoassay in 7 of 14 cases with MS and 1 case of subacute sclerosing panencephalitis, but not in IgG from noninflamed control tissue. This finding was confirmed with a solution-phase RIA, which measures higher affinity autoantibodies. These data demonstrate that autoantibodies recognizing MOG are present in substantially higher concentrations in the CNS parenchyma compared with cerebrospinal fluid and serum in subjects with MS, indicating that local production/accumulation is an important aspect of autoantibody-mediated pathology in demyelinating CNS diseases. Moreover, chronic inflammatory CNS disease may induce autoantibodies by virtue of epitope spreading.

Chemiluminescence immunoassay for chloramphenicol

In recent years, various chemiluminescent clinical immunoassay kits have been widely applied to the detection of hormones. However, a kit for chloramphenicol (CAP) is often absent from most commercial product lists, even though it is important to control the levels of CAP residues in foodstuffs too. Therefore, we describe a simple, solid-phase chemiluminescence immunoassay (CLIA) for the measurement of CAP in foodstuffs. A rabbit anti-CAP IgG is passively adsorbed onto the walls of polypropylene plates. The labeled antigen is horseradish peroxidase (HRP) conjugate of CAP. Luminol solution is used as the substrate of HRP. The light yield is inversely proportional to the concentration of CAP. The method has a similar sensitivity (0.05 ng/ml), specificity, precision, and accuracy to a conventional enzyme immunoassay (EIA). The intra-assay and inter-assay CVs of ten samples were <8% and <20%, respectively, and the analytical recovery of the method was 87-100%. The experimental correlation coefficient of dilution was found to be 0.999 using milk supernatant as buffer. The detection limit for the method was 0.1-10 ng/ml, and it displayed good linearity.

Development and comparison of two immunoassays for the detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance

This paper describes the development and comparison of two immunoassays for the detection of 2,4,6-trinitrotoluene (TNT) based on competitive inhibition. Two polyclonal antibodies were used in the immunoassay; one was prepared from 2,4,6-trinitrophenol–bovine serum albumin conjugate (anti-TNP-BSA Ab, goat IgG) and another from 2,4,6-trinitrophenyl–keyhole limpet hemocyanine conjugate (anti-TNPh-KLH Ab, rabbit IgG). A solid-phase immunoassay that consisted of physically immobilized TNP-BSA conjugate was allowed to interact with these antibodies and the resonance angle changes due to the biomolecular interactions were monitored by surface plasmon resonance (SPR). The quantification of TNT was based on the change in the resonance angle shift for binding of antibodies with a TNP-BSA conjugate, which occurred in the presence of TNT in solution. Both antibodies showed a high degree of affinity for TNT and varying degrees of cross-reactivity to the related nitroaromatic derivative compounds, such as 2,4-dinitrotoluene (DNT), 1,3-dinitrobenzene (DNB), 2-amino-4,6-dintitrotoluene (2A-4,6-DNT), 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) and TNP. The anti-TNPh-KLH Ab prepared in our laboratory showed a detection limit of 0.006 ng/ml (6 ppt), which was comparable to or even better than the detection limit (0.095 ng/ml or 95 ppt) of a commercially available anti-TNP-BSA Ab for detection of TNT. A pepsin solution prepared with a glycine–HCl buffer (pH 2.0) was used for regeneration of the sensor surface. The duration for a single immunocycle was ∼19 and ∼13 min for anti-TNP-BSA Ab and anti-TNPh-KLH Ab immunosystems, respectively. The proposed immunoassay is simple and possesses good analytical characteristics for quantification of TNT.

Solid-Phase Immunoassays in Mechanism-Based Drug Discovery: Their Application in the Development of Inhibitors of the Molecular Chaperone Heat-Shock Protein 90

High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis.

Kinetics of protein binding in solid-phase immunoassays: Theory

In a solid-phase immunoassay, binding between an antigen and its specific antibody takes place at the boundary of a liquid and a solid phase. One of the reactants (receptor) is immobilized on a surface. The other reactant (ligand) is initially free in solution. We present a theory describing the kinetics of immunochemical reaction in such a system. A single essential restriction of the theory is the assumption that the reaction conditions are uniform along the binding surface. In general, the reaction rate as a function of time can be obtained numerically as a solution of a nonlinear integral equation. For some special cases, analytical solutions are available. Various immunoassay geometries are considered, in particular, the case when the reaction is carried out on a microspot.

Preanalytical Influences on DPC IMMULITE 2000 Intact PTH Assays of Plasma and Serum from Dialysis Patients

Measurement of intact parathyroid hormone (PTH) is essential to the diagnosis and management of metabolic bone disease (1), hypercalcemia, hypocalcemia (2), and renal osteodystrophy (3). The effects of specimen type, collection temperature, and storage temperature on the in vitro stability of PTH differ by method and platform (4)(5)(6)(7)(8)(9)(10). Characterization of preanalytical effects unique to each method, platform, and patient population is important to prevent potential clinical misclassification. The Diagnostics Product Corporation (DPC) IMMULITE 2000 intact PTH assay is a solid-phase two-site chemiluminescent immunoassay with a monoclonal mouse capture antibody and a polyclonal goat signal antibody conjugated to alkaline phosphatase. The effects of preanalytical factors on this assay have been partially investigated. Underfilling of EDTA-plasma tubes decreased measured PTH in samples from both healthy persons and those with chronic kidney disease (CKD) (11). Storage of SST serum samples for 3 days at room temperature decreased measured PTH in SST serum samples, an effect not seen with EDTA plasma (6). Results from EDTA plasma paradoxically increased after storage at 4 °C (12). Despite evidence that PTH is more stable in EDTA-anticoagulated specimens (5)(6)(7)(9)(10), the manufacturer describes an instability associated with EDTA-plasma samples and therefore recommends cold (2–8 °C) collection, centrifugation, and storage until analysis (13). At our institution, PTH is most frequently measured for management of CKD and helps direct therapies, including calcium, vitamin D, bisphosphonates, and parathyroidectomy. Preanalytical influences on PTH assays may be atypical in CKD because samples contain increased concentrations of N-terminal truncated PTH fragments that cross-react with intact PTH assays (14). Accordingly, we have investigated preanalytical variables in a dialysis-dependent CKD population. The study was approved by the St. Paul’s Hospital ethics committee. Five predialysis samples were …

Electrochemical Bioassay Utilizing Encapsulated Electrochemical Active Microcrystal Biolabels

A new approach to perform electrochemical immunoassay based on the utilization of encapsulated microcrystal was developed. The microcrystal labels create a "supernova effect" upon exposure to a desired releasing agent. The microcrystal cores dissolve, and large amounts of signal-generating molecules diffuse across the capsule wall into the outer environment. Layer-by-Layer (LbL) technology was employed for the encapsulation of electrochemical signal-generating microcrystals (ferrocene microcrystals). The encapsulated microcrystals were conjugated with antibody molecules through the adsorption process. The biofunctionalized microcrystals were utilized as a probe for immunoassays. The microcrystal-based label system provided a high-signal molecule to antibody (S/P) ratio of 10(4)-10(5). Microcrystal biolabels with different antibody surface coverage (1.60-5.05 mg m(-2)) were subjected to a solid-phase immunoassay for the detection of mouse immunoglobulin G (M-IgG) molecules. The microcrystal-based immunoassay for the detection of M-IgG performed with microcrystals having antibody surface coverage of 5.05 mg m(-2) showed a sensitivity of 3.93 nA microg(-1) L(-1) with a detection limit of 2.82 microg L(-1).

Solid‐Phase Immunoassays

Solid-phase quantitative immunoassays are some of the most commonly used diagnostic tests for both soluble antigen composition and the assessment of cellular functions. These immunoassays (e.g., ELISA, ELISPOT) provide a highly sensitive means to measure the presence of antigen in defined and homogeneous samples such as purified proteins in buffer, as well as in undefined heterogenous biological samples such as cell lysates, tissue culture supernatants, blood, and other clinical samples. The sensitivity of these assays can enable (under optimal conditions) detection of protein concentrations in the picogram range. A recent modification of the basic ELISA immunoassay takes advantage of the phenomenon of grating-coupled surface plasmon resonance (GCSPR) to provide a label-free real-time variant of this solid-phase immunoassay. Using GCSPR, similar assessments of antigen-antibody interactions can be done with smaller sample sizes and in a microarray format that enables the simultaneous measurement of large numbers of antibody/antigen interactions on the same sensor chip. These measurements allow for a highly refined and sensitive determination of the effects that toxins can have on biological systems, and they can be applied to a variety of immune and non-immune protein, cell, and tissue evaluations.