Sensitivity Of Antigen Detection Research Articles

Surface Grafted Antibodies: Controlled Architecture Permits Enhanced Antigen Detection

The attachment of antibodies to substrate surfaces is useful for achieving specific detection of antigens and toxins associated with clinical and field diagnostics. Here, acrylated whole antibodies were produced through conjugation chemistry, with the goal of covalently photografting these proteins from surfaces in a controlled fashion, to facilitate rapid and sensitive antigenic detection. A living radical photopolymerization chemistry was used to graft the acrylated whole antibodies on polymer surfaces at controlled densities and spatial locations by controlling the exposure time and area, respectively. Copolymer grafts containing these antibodies were synthesized to demonstrate two principles. First, PEG functionalities were introduced to prevent nonspecific protein interactions and improve the reaction kinetics by increasing solvation and mobility of the antibody-containing chains. Both of these properties lead to sensitive (pM) and rapid (<20 min) detection of antigens with this surface modification technique. Second, graft composition was tailored to include multiple antibodies on the same grafted chains, establishing a means for simultaneously detecting multiple antigens on one grafted surface area. Finally, the addition of PEG spacers between the acrylate functionality and the pendant detection antibodies was tuned to enhance the detection of a short-half-life molecule, glucagon, in a complex biological environment, plasma.

DETECTION OF HISTOPLASMA CAPSULATUM ANTIGEN IN BRONCHIAL ALVEOLAR LAVAGE SPECIMENS BY IMPROVED ANTIGEN DETECTION ENZYME-LINKED IMMUNOASSAY

PURPOSE: Detection of H. capsulatum antigen by Enzyme-linked Immunoassay (EIA) is well documented, and an improved 2nd generation assay exhibits increased sensitivity and specificity for urine and serum specimens as compared to the original EIA. The purpose of this study is to compare sensitivity for detection of antigen in bronchial alveolar lavage (BAL) specimens in the 2nd generation vs. original H. capsulatum antigen assay.

Single-Chain Fragment Variable Antibody Piezoimmunosensors

In this paper, we describe a novel nonlabeled biosensor with high diagnostic potential for rapid and sensitive detection of antigens in complex biological samples. The biosensor comprises a piezoimmunosensor (PZ) displaying a specially constructed recombinant antibody on its surface. The recombinant single-chain fragment variable (scFv) antibody contained a cysteine within the linker amino acid sequence used to join the scFv variable heavy and light chains. The presence of cysteine induced the scFv construct to self-assemble as a densely packed rigid monolayer on the gold surface of a quartz crystal microbalance. scFv molecules in this self-assembled monolayer (SAM) exhibited a defined orientation and high areal densities, with scFv-modified microbalance surfaces displaying 35 times as many variable antigen-binding sites per square centimeter as surfaces modified with whole antibody. Experimental data show that the scFv SAM PZ is superior to Fab fragment, Fab fragment containing a free sulfhydryl group (i.e., Fab-SH), and whole antibody PZs regarding sensitivity and specificity. Because of their small uniform size (MW approximately 27000) and the ease with which they can be modified using genetic engineering, scFv's have significant advantages over whole antibodies in microbalance biosensor systems. We demonstrate here that the use of scFv containing a cysteine within the scFv linker sequence (i.e., scFv-cys) for preparation of biosensor surfaces markedly increases the density of available antigen-binding sites, yielding a system that is highly selective, rapid, and capable of detecting low concentrations of antigens in complex samples.

Community-acquired respiratory viruses

Community-acquired respiratory viruses

Applications of flow cytometry and immunohistochemistry to diagnostic hematopathology.

Diagnostic hematopathology depends on the applications of flow cytometric immunophenotyping and immunohistochemical immunophenotyping combined with the cytomorphology and histologic features of each case. Select cases may require additional ancillary cytogenetic and molecular studies for diagnosis. The purpose of this review is to focus on the applications of flow cytometric and immunohistochemical immunophenotyping of paraffin-embedded tissue to diagnostic hematopathology. Advantages and disadvantages of these techniques are examined. The literature is extensively reviewed (PubMed 1985-2003) with an emphasis on the most recent applications and those that are most useful clinically, both diagnostically and prognostically. Study Selection.-Studies were selected based on statistically significant results in large studies with reported adequate clinical follow-up. The methodology was reviewed in the selected studies to ensure reliable comparison of reported data. Flow cytometric immunophenotyping offers the sensitive detection of antigens for which antibodies may not be available for paraffin immunohistochemical immunophenotyping. However, paraffin immunohistochemical immunophenotyping offers preservation of architecture and evaluation of expression of some proteins, which may not be available by flow cytometric immunophenotyping. These techniques should be used as complimentary tools in diagnostic hematopathology. There are extensive applications of flow cytometric and immunohistochemical immunophenotyping to diagnostic hematopathology. As cytogenetic and molecular findings evolve in diagnostic hematopathology, there may be additional applications of flow cytometric and immunohistochemical immunophenotyping to this field of pathology.

Immunohistochemistry used as a screening method for persistent bovine viral diarrhea virus infection.

Cattle persistently infected (PI) with bovine viral diarrhea virus(BVDV) are a major source of infection to herds. To successfully control BVDV, it is necessary to identify and cull those cattle PI with BVDV. Immunohistochemistry (IHC) is a useful tool for sensitive and specific detection of BVDV antigens in infected cattle. Skin of cattle PI with BVDV is one of the tissues where BVDV can be consistently identified by IHC and is readily accessible for sampling. Use of IHC on skin biopsies (in the form of ear notches)as a method to identify cattle PI with BVDV has resulted in a reliable, affordable technique for mass testing of cattle at an early age without maternal antibody interference. The ability to test large numbers of cattle to identify those Pl with BVDV will enable implementation of programs for control and eventual eradication of BVDV.

T cell adhesion lowers the threshold for antigen detection.

Antigen detection and initiation of TCR signaling only occur, under physiological conditions, when T cells are adherent, and not in suspension. We show here that T cell adhesion causes an increase in the Ca(2+) content of intracellular stores and of the amount of phosphatidylinositol 4,5-bisphosphate in the plasma membrane, and enhances TCR-induced Ca(2+) signaling. This priming can be observed in freshly isolated T cells, in activated T cells, and in some T cell lines. Stimulation of T cells by specific monomeric MHC-peptide complexes only triggers Ca(2+) responses after T cell adhesion. This solves a controversial issue concerning the minimum valency of activatory TCR ligands. Adhesion-induced T cell priming not only occurs upon binding to artificial substrates such as immobilized ligands, but also upon interaction with dendritic cells. Thus, this phenomenon is likely to contribute to the high sensitivity of antigen detection by T cells in secondary lymphoid organs.

A Novel Antigen Detection Immunoassay for Field Diagnosis of Hepatitis C Virus Infection

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (˜5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.

Fabrication of Protein Microarrays for Immunoassay Using the Electrospray Deposition (ESD) Method.

In recent years new methods for active protein film fabrication have been rapidly developing. One of these methods is the electrospray deposition (ESD) method. This method allows the spontaneous deposition of many identical dots and has a remarkable spatial resolution as well as the ability to overcome limitations like low rates of deposition, low efficiency of substance transfer, and cross-contamination. In this study, an antibody-based protein array for high-throughput immunoassay was fabricated with the ESD method using a quartz mask with holes made by an abrasive jet technique. An antibody solution was electrosprayed onto an ITO glass and then deposited antibodies were cross-linked with a vapor of glutaraldehyde. The diameter of the spots was approximately 150 μm. The arrays were then incubated with corresponding target antigenic molecules and washed. The captured antigens were collectively detected by fluorescence and chemiluminescence. These signals were quantitatively visualized with a high-resolution charge-coupled device (CCD) detector. Sensitive (~1 ng/ml) and simultaneous detection of various antigens could be performed by enzyme linked immunosorbent assay or fluorescence immunoassay using this system.

Antigenic characterization of a marine fish iridovirus from grouper, Epinephelus spp

Iridoviruses, recognized as causative agents of serious systemic diseases, have been identified from more than 20 fish species. Antigenic properties of a pathogenic iridovirus isolated from grouper, Epinephelus spp., in Singapore (SGIV) were investigated using rabbit IgG against the virus. Antisera were prepared by immunization of rabbit with purified virions. The rabbit IgG was purified from antiserum using a protein A-agarose column and adsorbed onto acetone-dried grouper (GP) cells. The viral surface-exposed antigens were visualized by a combination of immunogold transmission electron-microscopy and by indirect immunofluorescence, and the viral antigenic related proteins were discriminated by Western blot. The cross immunofluorescence assay showed that the grouper virus isolate was serologically close to viruses of the genus Ranavirus of family Iridoviridae . The viral antigens were detected from virus infected-cell cultures as early as 4 h of post infection using IFAT, and could be detectable in virus-infected fish blood as early as 3 days post infection. Immuno-dot assays revealed that the rabbit anti-SGIV IgG allowed sensitive detection of SGIV viral antigens. This study will facilitate the development of diagnostic techniques and vaccines for grouper iridovirus.

Detection of Clostridium botulinum neurotoxin type A using immuno-PCR.

An immuno-polymerase chain reaction (immuno-PCR) has been developed for the sensitive detection of antigens, which greatly extends the detection limits of immunoassays. In the current study, the method was applied to the detection of Clostridium botulinum neurotoxin type A (BTx-A). Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte were demonstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) exceeded the conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) mol) by a 1000-fold enhancement in detection sensitivity. Detection of BTx-A antigens by immuno-PCR demonstrated 100% sensitivity and 100% specificity in 100-fold magnitude below the detection limit of ELISA. It is concluded that the immuno-PCR method could be used to detect a very low level of BTx-A for clinical diagnosis.

Disposable, low cost, silver-coated, piezoelectric quartz crystal biosensor and electrode protection.

The use of a commercial, silver-coated, piezoelectric quartz crystal as a disposable, low cost and reliable immunosensor is presented. The protection of the silver electrode from undesirable oxidation was achieved by polystyrene or carboxy-poly(vinyl chloride) (PVC-COOH) modification. In addition to serving as protection for the electrode, polymer films provided a substrate for antibody immobilization by either physical adsorption or covalent linkage. Polystyrene modification showed an additional advantage of improvement of surface smoothness. The atomic force microscope (AFM) and scanning electron microscope (SEM) were used to evaluate the morphologies of polymer films obtained by dip or drop coating techniques. It was found that drop coating provided more significant improvement in surface smoothness than dip coating, and the resulting sensor surfaces were more suitable for in situ liquid phase assay. Although PVC-COOH-modified sensors were not suitable for liquid phase assay because of the high surface roughness, the covalent linkages (amide bonds) between antibodies and -COOH groups in the polymer film offered better sensor performance in ex situ assay in terms of a higher antibody binding capacity and better antigen detection sensitivity.

Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry

Flow cytometry is the method of choice for the analysis of single cells with respect to the expression of specific antigens. Antigens can be detected with specific antibodies either on the cell surface or within the cells, after fixation and permeabilization of the cell membrane. Using conventional fluorochrome-labeled antibodies several thousand antigens are required for clear-cut separation of positive and negative cells. More sensitive reagents, e.g., magnetofluorescent liposomes conjugated to specific antibodies permit the detection of less than 200 molecules per cell but cannot be used for the detection of intracellular antigens. Here, we describe an enzymatic amplification technique (intracellular tyramine-based signal amplification, ITSA) for the sensitive cytometric analysis of intracellular cytokines by immunofluorescence. This approach results in a 10 to 15-fold improvement of the signal-to-noise ratio compared to conventional fluorochrome labeled antibodies and permits the detection of as few as 300–400 intracellular antigens per cell.

A novel signal amplification technology for ELISA based on catalyzed reporter deposition. Demonstration of its applicability for measuring aflatoxin B 1

In an earlier communication we have described a novel signal amplification technology termed Super-CARD, which is able to significantly improve antigen detection sensitivity in conventional Dot-ELISA by approximately 10 5-fold. The method utilizes hitherto unreported synthesized electron rich proteins containing multiple phenolic groups which, when immobilized over a solid phase as blocking agent, markedly increases the signal amplification capability of the existing CARD method (Bhattacharya, R., Bhattacharya, D., Dhar, T.K., 1999. A novel signal amplification technology based on catalyzed reporter deposition and its application in a Dot-ELISA with ultra high sensitivity. J. Immunol. Methods 227, 31.). In this paper we describe the utilization of this Super-CARD amplification technique in ELISA and its applicability for the rapid determination of aflatoxin B 1 (AFB 1) in infected seeds. Using this method under identical conditions, the increase in absorbance over the CARD method was approximately 400%. The limit of detection of AFB 1 by this method was 0.1 pg/well, the sensitivity enhancement being 5-fold over the optimized CARD ELISA. Furthermore, the total incubation time was reduced to 16 min compared to 50 min for the CARD method. Assay specificity was not adversely affected and the amount of AFB 1 measured in seed extracts correlated well with the values obtained by conventional ELISA.

A novel signal amplification technology based on catalyzed reporter deposition and its application in a Dot-ELISA with ultra high sensitivity

A novel strategy to improve significantly antigen detection sensitivity of Dot-ELISA by catalyzed reporter deposition (CARD) method of signal amplification has been developed. The method, termed Super-CARD, utilizes synthesized electron rich proteins having multiple binding sites as blocking agents. After completion of conventional Dot-ELISA, the solid phase bound horseradish peroxidase (HRP) oxidises the added labeled substrate, which deposits onto the solid phase. This deposition is markedly increased in the presence of immobilized electron rich proteins, which not only amplifies the signal but also increases the sensitivity. The high specificity of the amplification reaction avoids the generation of any false positive signal. The extremely high sensitivity of Super-CARD technology permits visual detection of as few as 800 rabbit IgG molecules (1.33×10 −21 mol). The method is approximately 10 5-fold more sensitive than conventional Dot-ELISA. Direct comparison with existing CARD methods demonstrates approximately 1.6×10 4-fold enhancement in detection sensitivity which is much higher than that of any other existing methods. The Super-CARD technology is specific, flexible and may be applied to clinical diagnostics.

Immunomagnetic concentration of antigens and detection based on a scanning force microscopic immunoassay

Scanning Force Microscopy has already been shown to be a convenient and rapid method for sensitive antigen detection and quantification. Here, we describe different improvement steps brought to a TSH Scanning Force Microscopic ImmunoAssay (SFMIA), each of them aiming to solve a previous limitation of the solid phase test format and leading to a significant sensitivity enhancement. First, superparamagnetic nanoparticles conjugated to monoclonal anti αTSH antibodies were used for the specific TSH capture step. Their magnetic properties allowed easy separation of the complexes obtained from relatively large reaction volumes by application of a High Gradient Magnetic Field System. As a consequence, complex formation could proceed in a stirred solution, which greatly enhances binding rates compared to previous `static' conditions of solid-phase reactions. It was established that, despite their small size, magnetic complexes could be moved over short distances by a NdFeB magnet magnetic field. This property was exploited to overcome diffusion barrier and boundary layer constraints and to drive magnetic complexes through the liquid, towards anti-βTSH antibodies immobilized on silica wafers. Finally, we significantly increased the complex number/surface area by a stepwise reduction of the biospecific solid phase area. The proposed steps permitted a 3-fold improvement in the TSH SFMIA dynamic range. Moreover, as little as 0.02 pg/ml (0.1 nIU/ml or 0.8 amol/ml) of TSH could be detected using 1 ml sample volumes. This is over 100 times more sensitive than the current performance of commercialized automated systems.

Detection of Cell and Tissue Surface Antigens Using Up-Converting Phosphors: A New Reporter Technology

A novel luminescent reporter for the sensitive detection of antigens in tissue sections or on cell membranes is described. It consists of submicron-size phosphor crystals (0.2–0.4 μm), which are surface labeled with avidin or antibodies and capable of binding specifically to antigens on intact cells or in tissue sections. These phosphor reporters exhibit two-photon, anti-Stokes luminescence by up-converting infrared to visible light and are named Up-converting Phosphor Technology (UPT). They typically consist of yttriumoxysulfides doped with two different lanthanides exhibiting photostable, strong emission in the visible (blue, green, and red) upon excitation in the infrared. This report describes the conjugation of phosphor particles to NeutrAvidin with the subsequent use of this conjugate in a model system consisting of prostate-specific antigen in tissue sections and the CD4 membrane antigen on human lymphocytes. An epi-illumination fluorescence microscope was adapted to provide near-IR excitation using a xenon lamp for visualization of the visible emission. Advantages of UPT are (i) permanent, strong, anti-Stokes emission of discrete wavelengths; (ii) unmatched contrast in biological specimens due to the absence of autofluorescence upon excitation with IR light; (iii) simultaneous detection of multiple target analytes; and (iv) low-cost microscope modifications. The new methodology has not only high potential value in diagnostic pathology as described here, but may offer advantages for the detection of proteins or nucleic acids when applied to molecular biology, genomic research, virology, and microbiology.

A highly sensitive immunofluorescence procedure for analyzing the subcellular distribution of GABAA receptor subunits in the human brain.

We designed a protocol to improve the immunohistochemical analysis of human brain structures, which overcomes the limited detection sensitivity, high background, and intense autofluorescence commonly associated with human tissue. This procedure was evaluated by using antibodies against major GABAA receptor subunits (alpha1, alpha2, alpha3, gamma2) in autopsy and surgical specimens. Tissue blocks were briefly fixed by immersion and pretreated with microwave irradiation in sodium citrate buffer. Immunoperoxidase staining revealed a marked enhancement of cell surface immunoreactivity and reduction of background in microwave-irradiated tissue, irrespective of its origin. For confocal laser scanning microscopy, immunofluorescence staining was optimized with the tyramide signal amplification (TSA) technique. This procedure not only dramatically increased the sensitivity for antigen detection but also totally suppressed autofluorescence, thus revealing the cellular and subcellular distribution of GABAA receptor subunits. A distinct neuron-specific expression pattern of the alpha-subunit variants was observed in cerebral cortex and hippocampal formation, along with widespread expression of the gamma2-subunit. Of particular interest was the prominent alpha2- and alpha3-subunit staining on the initial axon segment of pyramidal neurons. This protocol represents a major improvement for high-resolution studies of human brain tissue aimed at investigating morphological alterations underlying neurological diseases.

Construction of chimeric proteins between protein G and fluorescence-enhanced green fluorescent protein, and their application to immunoassays

Chimeric proteins consisting of an IgG-binding domain of protein G (PG) and a fluorescence-enhanced green fluorescent protein variant (EGFP) were constructed to evaluate their utility in fluoroimmunoassays (FIAs). Four chimeric proteins (PG-EGFPs) with an amino acid residue of Ala, Pro, Ser or Thr as the linker between the two domains were constructed and expressed in E. coli. They were efficiently expressed and could be purified to homogeneity with one-step IgG-affinity chromatography. The proteins retained similar fluorescence spectra to those of EGFP, and the binding constants, as evaluated using BIAcore, revealed their high affinity for rabbit IgG, goat IgG, sheep IgG and mouse IgG 1. PG-EGFP with Ala, Pro, Ser as the linker showed a high affinity for rabbit IgG while that with Thr had a 3-fold lower binding constant. When PG-EGFPs were applied to sandwich FIAs, a minimum detection limit of 0.1 μg/ml of BSA using anti-BSA antibody, or 1 μg/ml of rabbit IgG was achieved using microplate format. This detection sensitivity was comparable to that obtained using FITC-labeled secondary antibodies. Western blotting of hen egg lysozyme and BSA using PG-EGFPs was also possible with similar sensitivity. These data show the applicability of these easily prepared PG-EGFPs to various FIAs for sensitive detection of antigens.

Enhancement of immunohistochemical detection of HIV-1 p24 antigen in brain by tyramide signal amplification

Human immunodeficiency virus type 1 (HIV-1) infection of the brain has been demonstrated in formalin fixed, paraffin embedded post-mortem brain tissue (PM) by chromogenic immunohistochemistry for the HIV p24 antigen. The sensitivity of antigen detection is increased significantly by tyramide signal amplification (TSA) compared to the conventional peroxidase labelled Avidin–Biotin complex (ABC) technique. The TSA method also permitted the use of a lower concentration of primary antibody than is conventionally used. Sensitivity was enhanced further by microwave irradiation of the paraffin embedded tissues in citrate buffer. HIV-1 p24 antigen was also detected in PM brain tissue by TSA enhanced immunofluorescence and demonstrated increased sensitivity compared to the conventional immunofluorescence technique with a greatly reduced autofluorescence background.