Antigen-capture Immunoassay Research Articles

Analysis of Filovirus Entry into Vero E6 Cells, Using Inhibitors of Endocytosis, Endosomal Acidification, Structural Integrity, and Cathepsin (B and L) Activity

Ebola and Marburg viruses are believed to enter host cells by receptor-mediated endocytosis. The process has been studied through the use of inhibitors that affect host cell properties and recombinant pseudotyping systems in which filovirus structural glycoproteins mediate entry of foreign virus particles. The aim of the present study was to determine the effects of such treatments on the entry of wild-type filoviruses. Vero E6 cells were exposed to various inhibitors before, during, and after infection with filoviruses. Infected cultures were harvested early (18-24 h) and late (72 h) after infection, and effects of treatment on entry were measured by fluorescent antibody staining of cells or by antigen capture immunoassays, respectively. These prelimary results suggest that filoviruses enter host cells through receptor-mediated endocytosis via clathrin-coated pits and caveolae, that actin filaments and microtubules are important in the entry process, and that proteolytic digestion of glycoprotein 1 by endosomal proteases facilitates entry. These observations obtained using wild-type viruses confirm the results of studies utilizing recombinant systems and offer additional insights into filovirus entry.

Commercial Immunoassay Kits for the Detection of Influenza Virus Type A: Evaluation of Their Use with Poultry

Five antigen capture immunoassay test kits, Directigen Flu A (Becton Dickinson), QuickVue Influenza test kit (Quidel), FLU OIA (ThermoBiostar), Zstat Flu (ZymeTx, Inc.) and NOW FLU A Test (Binax) were used to detect avian influenza virus (AIV) in clinical specimens as per manufacturers' protocols. Each kit was shown to be specific for AIV propagated in embryonating chicken eggs (ECE); other respiratory viruses of poultry tested gave negative results. The Directigen Flu A kit proved to be 10-fold more sensitive than the other kits, capable of detecting 10(4.7) mean embryo lethal dose (ELD50)/ml in allantoic fluid; this is more sensitive than the hemagglutination test using chicken erythrocytes. None of the kits proved to be sufficiently sensitive to reliably detect AIV in oropharyngeal and cloacal swabs collected from chickens experimentally infected with AIV subtype H6N2. In two different experiments, individual swabs and pools of five or six swabs were tested. By virus isolation, 39 individual oropharyngeal swabs tested positive for AIV, but Directigen and Flu OIA only detected 2/39 and NOW FLU A 1/39. Zstat and QuickVue did not detect any. Five individual cloacal swabs positive by virus isolation were negative with all five kits. In a second experiment using pools of five swabs, 26 swab pools were positive by virus isolation and 5/26 were positive by Directigen, the only kit to provide any positive results. Five cloacal swab pools were also positive by virus isolation and 1/5 was positive by Directigen; all other test kits were negative. All of these experiments were performed using the H6N2 subtype of AIV. The results are disappointing, as the kits have proven to be insensitive for detecting AIV when compared with the gold standard, virus isolation. This limits their use in diagnostic field investigations. Individual or groups of chickens could be assumed to be positive for AIV if positive by any of the kits, but a negative result with any of the kits would not prove that birds were AIV free.

Thomsen-Friedenreich and Tn Antigens in Nipple Fluid: Carbohydrate Biomarkers for Breast Cancer Detection

Novel biomarkers would facilitate early and accurate diagnosis of breast cancer. The Thomsen-Freidenreich (TF) and Tn antigens are aberrantly glycosylated carbohydrate cancer-associated antigens found in approximately 80% of adenocarcinomas. Both TF and Tn are expressed on cell-surface glycoproteins and glycolipids. Nipple aspirate fluid (NAF) is concentrated in secreted proteins and lipids from cells that give rise to cancer. The objective of this study was to determine if NAF from breasts with cancer contains elevated levels of TF and Tn compared with NAF from normal breasts. A sensitive and specific antigen capture immunoassay for TF and Tn detection in NAF was developed for this purpose. Fifty NAF samples, 25 from breasts with cancer and 25 from normal breasts, were examined. Antigen capture immunoassays were done on the samples using monoclonal antibodies that specifically recognized either TF or Tn antigen in NAF. These antibodies captured serially diluted NAF samples, and the concentration of TF or Tn was determined by comparing absorbance values against a standard curve generated from standard sources of TF or Tn. TF and Tn were detected in 19 of 25 and 20 of 25 NAF samples from breasts with cancer, respectively, compared with 0 of 25 and 1 of 25 NAF samples from breasts without cancer (P < 0.001 for both TF and Tn). In 92% of the cancerous breast NAF samples tested, either TF or Tn was found. Simultaneous measurement of TF and Tn in NAF may facilitate the noninvasive detection of breast cancer and warrants further study.

Rapid and sensitive detection of avian influenza virus subtype H7 using NASBA

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) is an isothermal technique allowing rapid amplification and detection of specific regions of nucleic acid from a diverse range of sources. It is especially suitable for amplifying RNA. A NASBA/ECL technique has been developed allowing the detection of RNA from avian influenza virus subtype H7 derived from allantoic fluid harvested from inoculated chick embryos and from cell cultures. Degenerate amplification primers and amplicon capture probes were designed enabling the detection of low and highly pathogenic avian influenza of the H7 subtype from the Eurasian and North American lineages and the Australian sub-lineage. The NASBA/ECL technique is specific for subtype H7 and does not cross-react with other influenza subtypes or with viruses containing haemagglutinin-like genes. The assay is 10- to 100-fold more sensitive than a commercially available antigen capture immunoassay system. The NASBA/ECL assay could be used in high throughput poultry screening programmes.

A sensitive immunoassay to detect the α-chemokine GRO in rabbit blood and lung fluids

GRO-α, GRO-β and GRO-γ are closely related peptides that stimulate growth of tumor cells and activate leukocytes in acute inflammatory reactions. In order to study the biology of GRO peptides in the lungs of experimental animals, we have developed and characterized a sensitive and specific immunoassay for rabbit GRO, and used this assay to measure GRO in rabbit lung fluids and plasma. GRO was cloned from a rabbit cDNA library and expressed in Escherichia coli. Specific goat polyclonal antibodies were used to create an antigen-capture immunoassay. The assay is sensitive to approximately 30 pg/ml GRO and does not crossreact with rabbit IL-8 or MCP-1, or human GRO. The assay accurately measures GRO in rabbit bronchoalveolar lavage fluid, plasma and serum. Rabbit erythrocytes bind little GRO and do not interfere with the detection of GRO in lung fluids. Circulating GRO was detected in the plasma of 4 of 6 pathogen-free rabbits, but the function of circulating GRO in normal animals is uncertain. This immunoassay will facilitate the study of the biology of GRO in rabbits with acute and chronic inflammation in the lungs and other tissues.

Reverse Transcriptase and Corresponding Activity-Blocking Antibody for Monitoring SIV℠Infection in Macaques

A nonradioactive reverse transcriptase (RT) assay was used to measure RT activity in serum during the viremia peak associated with primary infection and for measuring the generation and maintenance of RT activity-blocking antibody (RTb-ab) titers during and after seroconversion in SIV-infected macaques. The RT assay was compared to an antigen capture immunoassay designed for HIV-2/SIVsm and was found to be approximately 40 times more sensitive in detecting SIVsm in serum from infected macaques. The RT assay detected RT activity in serum corresponding to levels from 3 pg/ml. Earliest detection of viral replication using the RT assay was on day 6-8, with a peak at day 10 (up to 8000 pg/ml). The earliest detection of RTb-ab was seen on day 17-23, with established RTb-ab titers by day 29, followed by increasing titers of 15,000-120,000 by day 62-77. The usefulness of RT and RTb-ab for monitoring the course of SIV infection in monkey models is discussed.

Analysis of charge distribution of lambda- and kappa-IgA in IgA nephropathy by focused antigen capture immunoassay.

The finding that eluted mesangial IgA and serum IgA from patients with IgA nephropathy (IgAN) had a restricted anionic charge contrasting with normal serum IgA prompted us to examine the charge of kappa- and lambda-subclasses of IgA. Previous studies are not totally satisfactory because either total IgA without further dividing into subclass or only kappa- and lambda-IgA1 was examined. A new approach is described to study the electrostatic property of total IgA and its light chain subclasses. The new focused antigen capture immunoassay (FACIA) allows us to separate the immunoglobulins by isoelectric point, then to capture by the heavy chain class and finally to visualize according to the light chain class. This method works well with whole blood or serum without the need of prior purification by affinity chromatography. The serum total IgA and lambda-IgA levels in patients with IgAN were significantly higher than that of healthy controls. Elevated lambda-IgA levels in patients with IgAN resulted in reduced kappa/lambda ratio of total IgA. Similar to our previous findings in IgA1 with O-linked oligosaccharide side chain, lambda-IgA from patients with IgAN or health controls is highly anionic whereas kappa-IgA is relatively cationic. The higher anionic/cationic ratio observed in total IgA from patients compared with controls was due to the higher concentration of lambda-IgA from the former group. Raised anionic lambda-IgA in IgAN may be contributory to the immunopathogenesis through its selective mesangial binding.

Circulating anodic antigen (CAA) levels in different age groups in a Zimbabwean rural community endemic for Schistosoma haematobium determined using the magnetic beads antigen-capture enzyme-linked immunoassay.

A simplified version of the magnetic bead antigen-capture enzyme-linked immunoassay (MBAC-EIA) was used to detect circulating anodic antigen (CAA) in individuals of different age groups with Schistosoma haematobium infection only in an endemic area of Zimbabwe. An overall positive correlation between S. haematobium egg excretion and CAA levels was demonstrated. The age profile for CAA levels was generally comparable with the age profile of S. haematobium prevalence and intensity of infection. The CAA levels were higher in younger (5-14 years of age) individuals than in older (greater than 14 years of age) ones. Since the sensitivity of the MBAC-EIA in the diagnosis of S. haematobium infection was found to be 97%, CAA levels appear to be a useful indicator of worm burden in an endemic area.

Kinetics of serum levels of interleukin-6 in Staphylococcus aureus septicemia.

The kinetics of Il-6 serum concentrations were analyzed in 17 patients with a culture verified Staphylococcus aureus septicemia. The analyses were performed using an antigen capture immunoassay. All patients had elevated IL-6 serum concentrations on admission. In most cases a subsequent rapid decrease to low levels was registered within 10 days. The IL-6 serum concentrations reflected the clinical course. In sera sampled on admission a relationship was found between IL-6 serum concentrations and levels of C-reactive protein.

Monitoring Poultry Farms for Salmonella by Drag-Swab Sampling and Antigen-Capture Immunoassay

Drag-swab (DS) sampling, at the rate of four DS gauze pads per flock (house); modified culture procedures (novobiocin-supplemented plating media and delayed secondary selective enrichment); and Salmonella antigen-capture (SAC) technology were combined in screening one layer flock and 38 market-age broiler flocks. The results showed that low (negative) SAC sample-to-positive control (S/P) ratios were related to the negative culture recovery of Salmonella. Similarly, high (positive) S/P ratios were related to and indicative of positive culture recoveries. Extensive sampling and testing of 18 of the 39 flocks disclosed A) that five flocks with negative culture recoveries from feathers and freshly voided feces had essentially no positive DS-SAC values, and B) that 13 flocks with positive culture recoveries from feathers and/or fresh feces all had positive DS-SAC values.

Determination of human antibodies to specific antigens of cytomegalovirus using an antigen capture immunoassay

The antigen capture immunoassay which is described herein is based on the binding of specific antigens of cytomegalovirus (CMV) by monoclonal antibodies bound to a solid phase. The specificity of the binding was demonstrated by the analysis of antigens labelled with [ 35S]methionine and captured by the bound monoclonal antibodies. These specific antigens are recognized in turn by specific anti-cytomegalovirus antibodies in human sera. The immunoassay permits quantitation of these specific anti-cytomegalovirus antibodies and should facilitate both qualitative comparisons of the antibodies against specific CMV antigens in different individuals.

HIV Infection in 1968-Reply

In Reply. — The purpose of our Brief Report was to document human immunodeficiency type 1 (HIV-1) infection in 1968 and 1969 in the United States in a patient who, as we suggested earlier in 1984, 1 fulfilled the Centers for Disease Control diagnostic criteria for AIDS. We established HIV-1 infection by two specific, highly sensitive independent tests (Western blot and antigen capture immunoassays) and also attempted to eliminate artifacts due to storage through use of appropriate controls. Although it was not our purpose to review suspected AIDS cases in the pre-AIDS era, we did cite one review of the early literature back to 1902, 2 which antedated and overlapped the report of Huminer et al; the latter was not yet available to us at the time of submission of our article for publication. It is noteworthy that the referenced cases with positive serological tests for human immunodeficiency virus