D. Kornelis

Up-Converting Phosphor Technology-Based Lateral Flow Assay for Detection of Schistosoma Circulating Anodic Antigen in Serum

ABSTRACT Schistosoma sp. circular anodic antigen (CAA) serum concentrations reflect actual worm burden in a patient and are a valuable tool for population screening and epidemiological research. However, for the diagnosis of individual imported schistosomiasis cases, the current enzyme-linked immunosorbent assay (ELISA) lacks sensitivity and robustness. Therefore, a lateral flow (LF) assay was developed to test CAA in serum for individual diagnosis of imported active schistosome infections. Application of fluorescent submicron-sized up-converting phosphor technology (UPT) reporter particles increased analytical sensitivity compared to that of the standard ELISA method. Evaluation of the UPT-LF test with a selection of 40 characterized epidemiologic samples indicated a good correlation between signal intensity and infection intensity. Subsequently, the UPT-LF assay was applied to 166 serum samples of Dutch residents (immigrants and travelers) suspected of schistosomiasis, a case in which group routine antibody detection frequently fails straightforward diagnosis. The UPT-LF assay identified 36 CAA-positive samples, compared to 15 detected by CAA-ELISA. In conclusion, the UPT-LF assay is a low-complexity test with higher sensitivity than the CAA-ELISA, well suited for laboratory diagnosis of individual active Schistosoma infections.

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.

Mapping fucosylated epitopes on glycoproteins and glycolipids of Schistosoma mansoni cercariae, adult worms and eggs.

The developmental expression of the antigenic fucosylated glycan motifs Fucalpha1-3GalNAcbeta1-4GlcNAc (F-LDN), Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F), GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis X), and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) in Schistosoma mansoni cercariae, adult worms and eggs, was surveyed using previously defined anti-carbohydrate monoclonal antibodies (mAbs). Lewis X was found both on glycolipids and glycoproteins, yet with completely different expression patterns during the life-cycle: on glycolipids, Lewis X was mainly found in the cercarial stage, while protein-conjugated Lewis X was mainly present in the egg stage. Also protein-conjugated LDN-F and LDN-DF were most highly expressed in the egg-stage. On glycolipids LDN-DF was found in all three examined stages, whereas LDN-F containing glycolipids were restricted to adult worms and eggs. The motifs F-LDN and F-LDN-F were found both on glycoproteins and glycolipids of the cercarial and egg stage, while in the adult stage, they appeared to occur predominantly on glycolipids. Immunofluorescence assays (IFA) showed that these F-LDN and F-LDN-F containing glycolipids were localized in a yet undefined duct or excretory system of adult worms. Murine infection serum showed major reactivity with this adult worm duct-system, which could be fully inhibited by pre-incubation with keyhole limpet haemocyanin (KLH). Clearly, the use of defined mAbs provides a quick and convenient way to map expression profiles of carbohydrate epitopes.

Tools for diagnosis, monitoring and screening of Schistosoma infections utilizing lateral-flow based assays and upconverting phosphor labels

The potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosoma antibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of either Schistosoma mansoni or Schistosoma haematobium infections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against different Schistosoma species or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring.

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.

Application of the FITC-anti-FITC-gold system to ultrastructural localization of antigens.

We report the application of a fluorescein isothiocyanate (FITC)-anti-FITC method to localize antigens at the ultrastructural level. In the systems studied, the anti-FITC-based detection method displays high specificity and sensitivity. These observations, combined with ease of production and with availability of FITC-protein conjugates, suggest that the FITC-anti-FITC method is a good alternative to presently used methods and is widely applicable to immunochemical and immunocytochemical procedures. The same preparation and protocol can be used for light and electron microscopic studies, thereby reducing possible artifacts introduced if different procedures are used. In the present study, two systems were used to test the method. One system used an FITC-labeled monoclonal antibody (MAb) to schistosome circulating cathodic antigen. In this system, the label was detected in the gut of adult Schistosoma mansoni by an anti-FITC MAb conjugated to 10-nm gold particles. The second system used human IgM antibodies pooled from patients infected with Schistosoma mansoni. In this system detection was accomplished using an anti-human IgM-FITC conjugate followed by the anti-FITC-Au antibody conjugate.

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 Rossmans 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.

Schistosoma mansoni: Analysis of monoclonal antibodies reactive with gut-associated antigens

The analysis of a series of monoclonal antibodies (mAbs) developed in our laboratory against gutassociated antigens ofSchistosoma mansoni is described. It was found that mAbs that recognized epitopes of antigens in the gut and on the eggshell were mainly of the IgM isotype; these epitopes are likely to be carbohydrate in composition. Of a number of mAbs that were reactive with antigens important to the human humoral immune response, 75% appeared to be reactive with the circulating cathodic antigen.

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.

Immunodiagnosis of human schistosomiasis using different immunoprecipitation techniques.

SummaryOne hundred sera of individuals infected with Schistosoma mansoni and/or S. haematobium were examined for the presence of specific anti Schistosoma antibodies by means of different immunoprecipitation techniques: immunoelectrophoresis, immunodiffusion, immunoelectroosmophoresis (on two different supports), and electroimmunodiffusion. The immunoelectroosmophoresis proved to be superior to the other immunoprecipitation techniques, its main advantages being sensitivity, rapidity, and economic use of reagents. Precipitins against the antigen of the intermediate host, Biomphalaria glabrata, were demonstrated in 66% of the sera.