CD8 T cell autoreactivity to preproinsulin epitopes with very low human leucocyte antigen class I binding affinity.

Beta cells presenting islet epitopes are recognized and destroyed by autoreactive CD8 T cells in type 1 diabetes. These islet-specific T cells are believed to react with epitopes binding with high affinity to human leucocyte antigen (HLA) expressed on beta cells. However, this assumption might be flawed in case of islet autoimmunity. We evaluated T cell recognition of the complete array of preproinsulin (PPI) peptides with regard to HLA binding affinity and T cell recognition. In a comprehensive approach, 203 overlapping 9-10mer PPI peptides were tested for HLA-A2 binding and subjected to binding algorithms. Subsequently, a high-throughput assay was employed to detect PPI-specific T cells in patient blood, in which conditional HLA ligands were destabilized by ultraviolet irradiation and HLA molecules refolded with arrays of PPI peptides, followed by quantum-dot labelling and T cell staining. Analysis of patient blood revealed high frequencies of CD8 T cells recognizing very low HLA binding peptides. Of 28 peptides binding to HLA-A2, a majority was predicted not to bind. Unpredicted peptides bound mainly with low affinities. HLA binding affinity and immunogenicity may not correlate in autoimmunity. Algorithms used to predict high-affinity HLA peptide binders discount the majority of low-affinity HLA binding epitopes. Appreciation that peptides binding HLA with very low affinity can act as targets of autoreactive T cells may help to understand loss of tolerance and disease pathogenesis and possibly point to tissue-specific immune intervention targets.

A simple and rapid treatment (trichloroacetic acid precipitation) of serum samples to prevent non-specific reactions in the immunoassay of a proteoglycan.

In our laboratory serum components interfering in the immunoassay of the schistosome proteoglycan circulating anodic antigen (CAA) in serum necessitated us to develop a simple technique by which the non-specific reaction of negative control sera could be prevented. Trichloroacetic acid was added to serum samples to precipitate interfering (glyco-)proteins. After centrifugation, the supernatant was neutralized and used directly either in the ELISA or in the indirect haemagglutination. This method gave satisfactory results, i.e., negative control sera did no longer give false positive reactions, while the titre of the positive controls remained unaffected. This method could also be successfully applied for the pretreatment of urine samples.

Presence of the schistosome circulating anodic antigen (CAA) in urine of patients with Schistosoma mansoni or S. haematobium infections.

We investigated the presence of the circulating anodic antigen (CAA) in the urine of schistosomiasis patients. This genus specific antigen was hitherto demonstrated only in the serum of schistosomiasis patients. The urine of 80 patients with Schistosoma mansoni infections, 33 patients with S. haematobium infections, and 2 patients with mixed S. haematobium and S. mansoni infections were screened by a quantitative enzyme-linked immunosorbent assay (ELISA). CAA was demonstrated in 81% of those with intestinal schistosomiasis and in 97% of those with urinary schistosomiasis. CAA titers were less than 1:0.2-1:51.2. Results were compared with circulating cathodic antigen (CCA) titers in urine obtained in an indirect hemagglutination assay (IHA). CCA was generally not detectable in the urine of patients with S. haematobium infection, but was demonstrated in the urine of 85% of the patients with S. mansoni infection. Both CAA titers and CCA titers correlated positively with the number of S. mansoni eggs excreted in the feces, but CAA titers did not show a significant correlation with the number of S. haematobium eggs in urine. Both antigen titers showed a moderate correlation with the serum CAA level in schistosomiasis mansoni. The discovery of CAA in the urine of the majority of schistosomiasis patients tested suggests the use of urine samples for non-invasive immunodiagnosis of the disease.

Sensitive determination of circulating anodic antigen in Schistosoma mansoni infected individuals by an enzyme-linked immunosorbent assay using monoclonal antibodies.

From a panel of mouse monoclonal antibodies reactive with a repeating epitope of the schistosome circulating anodic antigen, an IgG1 monoclonal antibody was selected. This monoclonal antibody was applied in a sandwich enzyme-linked immunosorbent assay as capture antibody and as alkaline phosphatase labeled conjugate. This assay allowed a sensitive quantitation of circulating anodic antigen in serum samples of infected individuals, detecting less than 1 ng antigen/ml serum. In Schistosoma mansoni infected individuals from Zaire, the level of antigen in serum correlated with fecal egg output. The lower detection level of the immunoassay corresponded to a level of about 10 eggs/gm feces.

Quantitative determination of circulating antigens in human schistosomiasis mansoni using an indirect hemagglutination assay.

In serum and urine specimens collected from a group of Schistosoma mansoni infected individuals from Makundju, Zaire, the schistosome circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA) were quantitatively determined using an indirect hemagglutination reaction with sheep erythrocytes sensitized with mouse IgM monoclonal antibodies directed against these circulating antigens. Levels of CAA in serum (up to 5 ng/ml) and CCA in serum and urine (up to 50 ng/ml) were strongly correlated with egg excretion and with each other. No correlation was found between egg excretion and antibody levels against the circulating antigens. Antigen was detectable only in patients excreting greater than 500 eggs per gram of feces.

Recognition of gut-associated antigens by immunoglobulin M in the indirect fluorescent antibody test for schistosomiasis mansoni.

The specific reactivity of immunoglobulin M antibodies, as measured by a commonly used immunodiagnostic technique for schistosomiasis mansoni, the immunofluorescence assay (IFA) on Rossman-fixed sections of adult worms, was analyzed with monoclonal antibodies. Using monoclonal antibodies directed against 3 major gut-associated antigens, circulating anodic antigen (CAA), circulating cathodic antigen (CCA) and the 32 kDa antigen in blocking experiments, it was demonstrated that the measured IFA reaction was primarily due to reactivity with CCA. After blocking with anti-CCA monoclonal antibody, a mean decrease of fluorescent antibody titres of 95.8%, 92.9% and 83.8% was observed for sera from groups of patients with a recent infection (0-6 months), a static infection (6 months - 5 years), and a chronic infection (over 5 years), respectively. Titres of the group of recently infected patients were, after blocking, significantly lower (P less than 0.05) than those of the group of chronically infected patients.

Detection of the schistosome circulating cathodic antigen by enzyme immunoassay using biotinylated monoclonal antibodies.

We have developed an enzyme immunoassay (ELISA) for the quantification of the schistosome circulating cathodic antigen (CCA), a glycoprotein associated with the syncitium lining the gut of the parasite. A mouse monoclonal antibody of IgG3 isotype was used as coating (antigen-capture) antibody, while a biotinylated mouse monoclonal IgM was used as second (antigen-detecting) antibody. Streptavidin-alkaline phosphatase was used as enzyme label. The lower detection limit of the assay was 1.0 ng of the trichloroacetic acid soluble fraction of adult worm antigen (AWA-TCA) per ml, which corresponds to approximately 0.2 ng CCA per ml. The ELISA showed a linear range from 1.0 to 62.5 ng AWA-TCA per ml. Serum and urine samples of 16 individuals infected with Schistosoma mansoni (egg counts ranging from 5 to 4820 eggs per gram of faeces) were tested in the assay. Antigen titres ranged from less than 4-8192. This assay represents a considerable advantage in diagnosis of Schistosoma infections as it allows the detection and quantification of CCA in serum and urine in even lightly infected individuals.

Evaluation of an ELISA for combined measurement of CAA and CCA in schistosomiasis mansoni.

An enzyme-linked immunosorbent assay was developed for combined measurement of schistosome circulating anodic antigen (CAA) and circulating cathodic antigen (CCA). Monoclonal antibodies against CAA and CCA were used as coating and as fluorescein-labeled detecting antibodies in a FITC-anti-FITC system. The lower detection limit of the assay was 1.1 ng antigen (AWA-TCA)/ml. Serum samples of Schistosoma mansoni infected individuals from Zaire (n = 60) and Burundi (n = 60) were tested in this assay and in single-antigen ELISAs. Sensitivities of assaying for CAA, CCA, combined CAA + CCA, and of parallel testing for CAA and for CCA were calculated from titres and antigen concentrations. With serum samples from the heavily infected individuals (Zaire), all assays had a sensitivity of 97% or higher. In contrast, with serum samples from individuals from Burundi (low to moderate infections) it was shown that combined testing resulted in a slightly lower sensitivity than testing for individual antigens. By parallel testing for CAA and CCA, the sensitivity could be increased considerably (to 95%), however.

Enhanced detection of Schistosoma circulating antigens by testing 1 ml urine samples using immunomagnetic beads.

The potential advantage of increasing the sample volume for enhanced detection of two Schistosoma circulating antigens in urine has been evaluated. Two different, monoclonal antibody based, immunomagnetic beads assays detecting circulating anodic antigen (CAA) and circulating soluble egg antigen (CSEA), respectively, were investigated. By increasing the sample volume to 1.0 ml, the lower detection limit of antigen dilution series was improved six eight-fold in both immunomagnetic beads assays. Indeed, an improvement could be shown when testing the urine of Schistosoma-infected individuals with these magnetic beads assays. To compare the sensitivity of the immunomagnetic beads method with the standard ELISA, urine was experimentally adjusted to mirror low natural infection intensities. CSEA-positive urine samples were adjusted to levels below the detection limit of the corresponding ELISA and indeed were still found positive by the CSEA immunomagnetic beads assay. These results demonstrate that by using immunomagnetic beads, an assay method which can utilise larger sample volumes than an ELISA, enhanced detection can be achieved. This might be particularly valuable in areas with low prevalence of schistosomiasis or for the assessment of cure after chemotherapy.

Antigen-stimulated IL-4, IL-13 and IFN-gamma production by human T cells at a single-cell level.

To understand the intricate balance and the coordinate expression of the Th1 and Th2 cytokines following a natural mode of T cell triggering, antigen-stimulated IL-4, IL-13 and IFN-gamma production was studied in primary peripheral blood mononuclear cell cultures at a single-cell level. Cells from filariasis patients who respond to parasite antigen by producing not only IFN-gamma but also IL-4 and IL-13 were stimulated with Brugia malayi adult worm antigen and analyzed for co-expression of cytokines by intracellular staining. IL-4 and IL-13 were frequently co-expressed (54% of IL-4+ cells stained for IL-13 and 29% of IL-13+ cells expressed IL-4 at all time points), whereas IFN-gamma expression was totally segregated from both IL-4 and IL-13. These data indicate that in human peripheral T cells the co-expression of the dominant Th1 and Th2 cytokines within a single cell is a rare event and that IL-13 is clearly more frequently associated with a Th2 than a Th1 type response in primary T cell cultures.

Detection of IgM antibodies directed against the gut-associated circulating cathodic antigen in sera from Schistosoma mansoni infected patients. Development and comparison of three enzyme-linked immunoassays.

The majority of the human IgM antibodies detected with an immunofluorescence assay (IFA) on adult worms are directed against the gut-associated circulating cathodic antigen (CCA). In order to study this phenomenon further we developed and evaluated three related ELISA methods to specifically detect IgM antibodies against purified CCA. The assays employed: 1) direct coating of CCA, 2) indirect coating of CCA via a monoclonal antibody, and 3) IgM antibody-capture by rabbit anti-mu chain antibodies. Using a group of 46 positive sera, it was found that the three ELISA's and the IFA were significantly correlated. To discriminate between positive and negative sera we used a cut-off level of average reactivity + 3 standard deviations of 50 negative sera. False negative reactions were not found in any of the ELISA's, while both in the direct and indirect ELISA one false positive reaction occurred. For further studies or diagnostic use the antibody-capture ELISA is recommended.

Schistosoma: analysis of monoclonal antibodies reactive with the circulating antigens CAA and CCA.

Using spleen cells of mice infected or immunized respectively with cercariae or antigen preparations of Schistosoma mansoni, S. haematobium or S. japonicum monoclonal antibodies (mAbs) were produced against the schistosome gut-associated antigens CAA (circulating anodic antigen) and CCA (circulating cathodic antigen). Fusions nearly exclusively produced either anti-CAA (n = 25) or anti-CCA mAbs (n = 55) with a strong isotype restriction (IgM, IgG1 and IgG3) against both antigens, the majority of anti-CAA mAbs being IgG1 and the majority of anti-CCA mAbs being IgM. The mAbs, which on the basis of their selection were reactive with multiple carbohydrate epitopes of CAA or CCA, were applied in different immunological techniques including immunofluorescence, a dot immunobinding assay and immunoelectrophoresis to study the epitope repertoire. Anti-CAA mAbs were found to be reactive with 5 different epitopes, none of which occurred as multiple epitopes on eggs. Anti-CCA mAbs, on the other hand, recognized at least 10 different epitopes, while 44% of anti-CCA mAbs recognized epitopes common to the adult worm and the egg. Both CAA- and CCA-epitopes were found to be developmentally expressed at the level of the tegument in cercariae, schistosomula and 5-day-old lung worms, but in the adult worm were primarily found in the gut. Thus, the production of panels of mAbs has not only resulted in the selection of reagents optimally performing in diagnostic immunoassays, but also allowed a more detailed study of the epitope repertoire of these important schistosome antigens.