Surface antigens of a filarial nematode: analysis of adult Brugia pahangi surface components and their use in monoclonal antibody production

The surface antigens of adult worms of the filarial nematode Brugia pahangi have been investigated further by surface radioiodination and detergent solubilisation techniques. In addition to yielding new information on the distribution of antigenic components of this stage, detergent-solubilised molecules were used in both radiometric and enzyme-linked assays for human and mouse antibody. These assays were subsequently used in screening for monoclonal antibodies from hybrid cells derived from animals infected with living parasites and boosted with detergent-extracted antigen. Three monoclonal antibody-producing cell lines were isolated, with differing antigenic specificities: Bp-1, which binds a non-iodinatable antigen with high ELISA activity; Bp-2, which reacts with a determinant found on but not unique to the major surface Iodogen-labelled 29 kDa antigen; and Bp-3, which is specific for a minor antigen of 20 kDa revealed by Iodogen labelling.     

Enzyme-linked immunosorbent assay for the detection and identification of coxsackie B antigen in tissue cultures and clinical specimens

An enzyme-linked immunosorbent assay (ELISA) has been developed for the identification of Coxsackie B antigens. This assay was capable of identifying and distinguishing all six Coxsackie B serotypes at concentrations one hundredfold to ten thousandfold less than could be detected by complement fixation (CF) systems. In addition, the Coxsackie B ELISA correctly identified the presence of Coxsackie B antigen in 19 of 21 tissue culture fluids and five of nine rectal swab specimens. Two additional rectal swab specimens reacted with Coxsackie B antisera but could not be conclusively serotyped. Tissue culture fluids and rectal swab specimens containing other viruses such as ECHO virus, Coxsackie virus A, rhinovirus, rotavirus and Norwalk virus were consistently negative in the assay. The Coxsackie B ELISA offers potential for the rapid identification of Coxsackie B antigens in clinical specimens and tissue culture systems.     

A comparison of the IFA and the ELISA for the demonstration of antibodies against schistosome gut-associated polysaccharide antigens in schistosomiasis

The applicability of two immunodiagnostic techniques was studied for the detection of antibodies against schistosome gut-associated polysaccharide antigens in human schistosomiasis mansoni: the immunofluorescent antibody reaction (IFA) using Rossman's fixed paraffin sections of adult worms and the enzyme-linked immunosorbent assay (ELISA) with a trichloroacetic acid soluble fraction of total adult worm antigens (AWA-TCA).With the IFA, gut-associated polysaccharide antigens could be demonstrated with an anti-IgM conjugate in a high percentage of the sera tested, although false-negative reactions were occasionally recorded. The use of an anti-IgG conjugate resulted in the demonstration of antibodies against additional antigens in the parenchyma of the worm and on the tegument. Specific IgM antibodies were present in higher concentrations in the sera from children than in those from adults.Using AWA-TCA as the antigen preparation in the ELISA, only antibodies against the circulating anodic antigen (CAA) could be demonstrated. Pretreatment of the ELISA-plates with poly-L-lysine to couple AWA-TCA was not necessary. The ELISA was sucessfully applied with anti-Ig, anti-IgG and anti-IgM conjugates. With anti-Ig conjugate the test was very sensitive and gave less false-negative reactions than the IFA. There was a significant difference between Ig, IgG, and IgM titres of children and adults. The use of an immunogalactosidase assay with a fluorogenic substrate in the ELISA, resulted in a test which was able to detect antibodies at ten times higher dilutions than with the immunoperoxidase assay.This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases     

A competitive direct enzyme-linked immunosorbent assay for the rapid detection of deoxynivalenol: development and application in agricultural products and feedstuff

A competitive direct enzyme-linked immunosorbent assay (cd-ELISA) was developed for the rapid detection of deoxynivalenol (DON) in food and feedstuff. Polyclonal antibodies against DON were generated by immunizing rabbits with 3-HS-DON-BSA conjugates. In this assay, the sensitivity (measured as IC₅₀) and limit of detection (measured as IC₁₅) were 0.03 and 0.003?mg?kg^?1, respectively. A total of eight sample types (barley, wheat, oat, maize, rice, flour, milk and feedstuff) were chosen to evaluate the cd-ELISA assay performance. The sample could be directly detected after extraction and dilution with double-distilled water. The limit of detection in the samples was in the range of 0.15–0.48?mg?kg^?1. In the end, the spike and recovery results in both cd-ELISA and high-performance liquid chromatography (HPLC) were compared for assay validation. The recovery results ranged between 70% and 100%. A good correlation (R²?=?0.9613) between ELISA and HPLC was obtained.     

An enzyme immunoassay for the detection of staphylococcal protein A in affinity-purified products

Rabbit antiserum, specific for protein A from Staphylococcus aureus, was conjugated to alkaline phosphatase and used in a double antibody solid-phase enzyme immunoassay. The assay was developed to monitor eluate from a large-scale protein A-Sepharose affinity column used to purify monoclonal antibodies for human clinical trials. The assay detected soluble protein A in the presence of immunoglobulin at concentrations as low as 4 ng/ml. Analysis of the product purified by affinity chromatography revealed the presence of protein A at ng/ml concentrations. The assay developed here can provide a reliable and convenient method for detecting soluble protein A.     

The relationship between the binding of primary antibody to solid-phase antigen in microtiter plates and its detection by the ELISA

Radioiodinated monomeric and dimeric M315 (mM315 and dM315) prepared from BALB/c ascites fluid by gel filtration and affinity chromatography were used to study the relationship between primary antibody binding to solid-phase dinitrophenylated gelatin in microtiter ELISAs and its indirect detection by enzyme-antibody conjugates and complexes. The relationship between the amount of mM315 or dM315 which binds to dinitrophenylated gelatin to the amount added is linear over a nearly 3-log range with a slope of 1; the amount of M315 which binds in this linear range after 24 h represents all of the active antibody in the system. On a binding site basis, mM315 was inhibited by a significantly lower amount of dinitrophenyl-(DNP)glycine than was dM315. The indirect detection of bound M315 over the same 3-log range using ELISA yielded a sigmoidal titration curve which encompassed a short linear region that had a slope of 0.9 or less. Plateauing of the titration plots at high input of both mM315 and dM315 was shown to be progressively exaggerated in direct relationship to the size of the enzyme-antibody conjugate used for their detection. The data show that the upper region of the sigmoidal ELISA titration plot is the result of steric hindrance of the detection system. Through the combined use of an 131I-enzyme-antibody immune complex (EIC) detection system and 125I-M315, it was shown that the deviation from the linear binding of 125I-M315 observed during its indirect detection in the so-called linear region of the ELISA titration curve was the result of changing ratios of bound EIC: bound primary antibody, not altered enzymic activity.     

IgG against Plasmodium falciparum variant surface antigens and growth inhibitory antibodies in Mozambican children receiving intermittent preventive treatment with sulfadoxine-pyrimethamine

This study aimed to evaluate whether intermittent preventive treatment in infants with sulfadoxine-pyrimethamine (IPTi-SP) had an effect on the acquisition of IgG against Plasmodium falciparum variant surface antigens (VSA) and growth-inhibitory antibodies in Manhiça, Mozambique. In addition, we assessed factors affecting the magnitude of these responses and the association between antibody levels and protection against malaria.IgG to VSA expressed by MOZ2, R29 and E8B parasite isolates were measured in plasma samples collected at 5, 9, 12 and 24 months of age by flow cytometry. Growth-inhibitory antibodies in dialyzed plasmas using GFP-D10 parasites were measured by flow cytometry at 12 and 24 months.IPTi-SP did not significantly modify the levels of IgG against VSA nor the growth-inhibitory capacity of antibodies up to 2 years of age. Age but not previous episodes of malaria influenced the magnitude of these responses. In addition, anti-VSA IgG levels were 7% higher in children with current P. falciparum infection and were associated with neighborhood of residence. Children aged 24 months had 10% less parasite growth than those aged 12 months (95% CI 0.88-0.93, P < 0.0001). Growth-inhibitory antibodies correlated with levels of IgG against AMA-1, when evaluating the 10% (R² = 0.444, P = 0.049) and 20% (R² = 0.230, P = 0.037) highest inhibitory samples. None of the responses were associated with subsequent risk of malaria.In conclusion, IPTi-SP does not negatively affect the development of antibody responses thought to be major contributors to the acquisition of immunity to malaria in infancy.     

Ex vivo simulation of leukocyte function: stimulation of specific subset of leukocytes in whole blood followed by the measurement of function-associated mRNAs

In order to characterize a wide spectrum of leukocyte functions with clinically applicable procedures, 0.06 ml each of heparinized whole blood was stimulated in triplicate for 4 h with phytohemagglutinin (T cell stimulator), heat aggregated IgG (IgG Fc receptor stimulator), lipopolysaccharide (toll-like receptor (TLR)-4 stimulator), zymosan (TLR-2 stimulator), monoclonal antibody against T-cell receptor alpha/beta chain, recombinant interleukin-2, and solvent controls, then 32 different leukocyte function-associated mRNAs were quantified by the method reported previously (Mitsuhashi et al. Clin. Chem. 2006). Two control genes (beta-actin, beta-2-microglobulin) were not affected by these stimulations, whereas the induction of CCL chemokines-2, 4, 8, 20, CXCL chemokines-3, 10, interleukin (IL)-8 (markers of leukocyte accumulation/recruit), granzyme B, perforin 1, tumor necrosis factor superfamily-1, 2, 5, 14, 15, CD16 (markers of cell killing), IL10, transforming growth factor beta 1 (humoral factors of immune suppression), forkhead box P3, CD25, arginase (cellular markers of immune suppression), IL2, IL4, interferon-gamma, IL17 (markers of various subsets of T helper cells), granulocyte-macrophage colony-stimulating factor (marker of antigen presenting cells), immunoglobulin heavy locus (marker of B-cells), vascular endothelial growth factor (marker of angiogenesis), pro-opiomelanocortin (marker of local pain), and CD11a mRNA (marker of leukocyte adherence to endothelium) were identified by these stimulations. The blood volume in this assay was 1.44 ml, and 4 h’ incubation in whole blood was physiological. Using triplicate aliquots of whole blood for both stimulant and solvent control, statistical conclusion was drawn for each stimulant for each mRNA. The method introduced in this study will be a new paradigm for clinical cellular immunology.     

Role of oxidative stress,angiogenesis and chemo-attractant cytokines in the pathogenesis of ischaemic protection induced by remote ischaemic conditioning: Study of a human model of ischaemia-reperfusion induced vascular injury

Aims

We explored the effect of remote ischaemic conditioning (RIC) on endothelial function and on circulating mediators.

Beneficial effects of cannabinoids (CB) in a murine model of allergen-induced airway inflammation: role of CB1/CB2 receptors

The endocannabinoid system (ECS) consists of two cannabinoid (CB) receptors, namely CB₁ and CB₂ receptor, and their endogenous (endocannabinoids) and exogenous (cannabinoids, e.g. delta-9-tetrahydrocannabinol (THC)) ligands which bind to these receptors. Based on studies suggesting a role of THC and the ECS in inflammation, the objective of this study was to examine their involvement in type I hypersensitivity using a murine model of allergic airway inflammation. THC treatment of C57BL/6 wildtype mice dramatically reduced airway inflammation as determined by significantly reduced total cell counts in bronchoalveolar lavage (BAL). These effects were greatest when mice were treated during both, the sensitization and the challenge phase. Furthermore, systemic immune responses were significantly suppressed in mice which received THC during sensitization phase. To investigate a role of CB_1/2 receptors in this setting, we used pharmacological blockade of CB₁ and/or CB₂ receptors by the selective antagonists and moreover CB₁/CB₂ receptor double-knockout mice (CB₁^−/−/CB₂^−/−) and found neither significant changes in the cell patterns in BAL nor in immunoglobulin levels as compared to wildtype mice. Our results indicate that the activation of the ECS by applying the agonist THC is involved in the development of type I allergies. However, CB₁/CB₂ receptor-independent signalling seems likely in the observed results.     

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand alpha-galactosylceramide bound to mouse CD1d

The alpha-galactosylceramide (alpha-GalCer) known as KRN7000 remains the best studied ligand of the lipid-binding MHC class I-like protein CD1d. The KRN7000:CD1d complex is highly recognized by invariant natural killer T (iNKT) cells, an evolutionarily conserved subset of T lymphocytes that express an unusual semi-invariant T cell antigen receptor, and mediate a variety of proinflammatory and immunoregulatory functions. To facilitate the study of glycolipid antigen presentation to iNKT cells by CD1d, we undertook the production of mouse monoclonal antibodies (mAbs) specific for complexes of KRN7000 bound to mouse CD1d (mCD1d) proteins. Three such monoclonal antibodies were isolated that bound only to mCD1d proteins that were loaded with KRN7000 or closely-related forms of alpha-GalCer. These mAbs showed no reactivity with mCD1d proteins that were not loaded with alpha-GalCer, nor did they bind to complexes formed by loading mCD1d with the self-glycolipid and putative iNKT cell ligand isoglobotrihexosylceramide. These complex-specific monoclonal antibodies allow the direct detection and monitoring of complexes formed by the binding of KRN7000 and other alpha-GalCer analogues to mCD1d. The availability of these mAbs should facilitate a wide range of studies on the biology and potential clinical applications of CD1d-restricted iNKT cells.