Defined Antigen Substrate Spheres Research Articles

Schistosoma mansoni: Use of antigens from excretions and secretions in immunodiagnosis

The use of antigens from excretions and secretions (ESA) of Schistosoma mansoni in two immunodiagnostic tests, the enzyme-linked immunosorbent assay (ELISA), and the defined antigen substrate spheres (DASS) system, has been extensively investigated. In comparison with total adult worm antigens (AWA), the sensitivity of the DASS tests remained the same, while that of the ELISA increased slightly when ESA was used. For further analysis, the ESA preparation was fractionated according to molecular weight, by gel filtration. The humoral immune response of immunized rabbits, infected mice, and humans to each of these molecular-weight fractions was determined by incubating an equal, nonsaturating amount of each ESA fraction in a double-antibody sandwich system, using Sepharose beads as a carrier. The humoral immune response of rabbits immunized with ESA was primarily directed against antigens with molecular weight between 50,000 and 70,000. In contrast, immunoglobulins from sera of infected mice or humans, reacted well with antigens from a large molecular-weight range. Screening of a large number of sera for the presence of specific antibodies is most conveniently executed with tests in which antigens, instead of antibodies, are bound to a matrix. However, binding of antigens to Sepharose beads or polystyrene microtiter plates was shown to decrease considerably with decreasing molecular weight of the antigen. Therefore, of all ESA fractions, those containing the high-molecular-weight antigens (MW > 200,000) gave the most sensitive DASS and ELISA tests. These high-molecular-weight excretory and secretory antigens, in contrast to a total-worm homogenate, and excretory and secretory antigens with a molecular weight lower than 200,000, possessed a high specificity for S. mansoni. The specificity of the high-molecular-weight preparation was shown to be mainly due to the presence of the circulating anodic polysaccharide antigen, since removal of this antigen by immunoadsorption led to a considerable decrease in specificity.

Separation and comparative immunoassay (DASS, ELISA) with antigens from adult Schistosoma mansoni

Antigenic fractions were prepared from adult Schistosoma mansoni by homogenization and solubilization in phosphate buffer, and subsequent separation on Ultrogel AcA-44. The fractions were tested for antigenicity in two immunodiagnostic techniques, the defined antigen substrate spheres (DASS) system, and the enzyme-linked immunosorbent assay (ELISA), using serum from schistosomiasis patients. The results obtained in the two tests correspond to each other. Sera from 25 patients with proven schistosomiasis were tested with ELISA, using the antigen fractions. For all sera tested, the titres obtained, were dependent on the antigen fraction used. However, areas of high antigenicity were different for different sera. Antigens with molecular weight lower than 60,000, gave negative results for about 30% of the sera but those with molecular weight higher than 60,000 gave positive results for all sera. For about 60% of the sera, the sensitivity of ELISA increased when antigens of increasing molecular weight were used. Cross reactions with two other helminth infections occurred mainly with high molecular weight antigens.

The serodiagnosis of human hydatid disease: 2. Additional studies on selected sera using indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS)

Sixty-one serum samples selected on the basis of reactivity in the complement fixation (CF) and latex agglutination (LA) test, were further examined for sensitivity and specificity by indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS). Twenty sera from healthy Europeans and 48 samples from patients with either schistosomiasis or trichinosis were also tested. Comparable levels of sensitivity were found between the CF and LA positive sera and IHA, ELISA and DASS. Of the CF positive LA negative group of sera, many were positive by DASS but only a few reacted in IHA and ELISA. Some cross reactivity was also observed in the schistosomiasis sera tested by IHA and ELISA.

Comparison of activation and blocking procedures in the use of defined antigen substrate spheres (DASS) for quantitative and visual assessment of conjugate activity

The use of Sepharose beads in the defined antigen substrate sphere (DASS) immuno-fluorescent system has been restricted by the need for sophisticated equipment for fluorescence quantitation since reliable subjective reading is not possible. This paper describes a method for visual assessment of results in this system. Antigen is coupled to sodium metaperiodate activated cross-linked Sepharose 4B beads and blocked with rhodamine-BSA prior to reduction. This procedure eliminates the use of the hazardous cyanogen bromide activation process and is potentially suitable for use in both direct systems for evaluation of specificity and potency of fluorescent conjugates and in indirect systems for serological screening of sera for specific antibody.

Schistosoma mansoni: Antigenic characterization of malate dehydrogenase isoenzymes and use in the defined antigen substrate spheres (DASS) system

Immunoelectrophoresis of Schistosoma mansoni homogenates against mouse antisera resulted in only one precipitation line, which showed malate dehydrogenase activity. Immunoprecipitins against schistosomal malate dehydrogenase were also demonstrated in sera from individuals with schistosomiasis. Analysis by the double-diffusion method showed that malate dehydrogenase antigens in S. mansoni, S. haematobium, and S. bovis are immunologically indistinguishable. Immunoelectrophoresis of isolated mitochondrial and cytoplasmic malate dehydrogenase, showed that only the mitochondrial enzyme is able to form a malate dehydrogenase active precipitation line. Rabbit antisera directed against purified mitochondrial malate dehydrogenase showed a reaction with the enzyme as judge by immunoelectrophoresis. A purified mitochondrial malate dehydrogenase preparation, coupled to Sepharose 4B, was used in the defined antigen substrate spheres (DASS) test. Sera from experimentally infected mice contained considerably higher levels of antibodies against the mitochondrial malate dehydrogenase preparation than sera from infected individuals.

Schistosoma mansoni: Microfluorometric determination of circulating anodic antigen and antigen-antibody complexes in infected hamster serum

In this study the applicability of the defined antigen substrate spheres (DASS) system for the detection of circulating anodic antigen (CAA) in experimental Schistosoma mansoni infections in hamsters and mice is described. Using the DASS system as an antibody-antigen-labeled antibody technique, CAA could be detected both as a free circulating antigen and as the antigen-component of circulating antigen-antibody complexes.

Comparison of fluorescent antibody tests for tuberculosis and paratuberculosis with antigens coupled to insoluble spheres or taken up by macrophages

Sera of 106 cattle from farms with histories of Mycobacterium johnei infection and sera from 15 human tuberculous patients as well as a number of control sera were examined by means of two different fluorescent antibody tests (FAT) for the occurrence of antibodies against M. johnei and M. tuberculosis respectively. The antigens used were PPD johnin and PPD tuberculin. In the macrophage uptake FAT (MU/FAT) mouse macrophages after phagocytosis of the tuberculins served as the matrix; in the tests performed using the defined antigen substrate spheres (DASS) system, Sepharose beads activated by CNBr were used for the matrix. A good correlation was found between the results of the DASS/FAT and those of the MU/FAT, which is known to be a sensitive and specific test in the diagnosis of Johne's disease in cattle. It is suggested that the FAT, with utilization of the DASS system, might have good prospects for routine examination for antibodies against species of Mycobacterium.

Schistosoma mansoni: Comparison of the immunoperoxidase techniques, DASS and ELISA, for human diagnosis

A comparative study of the applicability of immunoperoxidase techniques for the immunodiagnosis of Schistosoma mansoni infections in humans was performed. The two immunoperoxidase techniques used were the defined antigen substrate spheres (DASS) system and the enzyme linked immunosorbent assay (ELISA) technique. The latter technique was used in a micro- and a macrosystem. The results were compared with those obtained with the indirect fluorescent antibody (IFA) technique on frozen sections of the adult parasites. ELISA and DASS proved to be at least as specific and sensitive as IFA and showed considerable advantages compared with IFA. Results of ELISA and DASS could readily be observed with the naked eye.

Microfluorometric evaluation of the specificity of fluorescent antisera against mouse immunoglobulins with the defined antigen substrate spheres (DASS) system

Highly purified MOPC-21 IgG 1, MOPC-173 IgG 2a, MOPC-195 IgG 2b, MOPC-104 E IgM, and MOPC-315 IgA paraproteins, heterogeneous mouse IgG, Fab and Fc fragments of heterogeneous IgG were prepared and coupled to Sepharose beads. These beads were then used as artificial substrates to test the specificity of fluorescent antisera against mouse immunoglobulins by microfluorometry. By comparing the visual evaluation of stained plasma cells and measurements on beads, the highest permissible percentage impurity in a conjugate was determined. It was 14% for a fluorescein iso thiocyanate (FITC) and 6% for a tetramethyl rhodamine iso thiocyanate (TRITC) conjugate. By application of these criteria, 1 out of 7 tested commercial antisera and 6 out of 8 conjugates prepared in this laboratory proved to be satisfactory. The most common impurities were anti-light chain antibodies, as revealed by their reaction with Fab. With the bead system, a good impression of the specificity of an antiserum can be obtained. It gives, however, only approximate information on whether the conjugate will cause a high background staining in the biological specimen.

Mikrofluorometrischer Nachweis von Thyreoglobulin-Antikörpern mit dem definierten Antigen-Substrat-System (DASS)1): Microfluorometric demonstration of thyroglobulin antibodies with the defined antigen substrate spheres (DASS) system

Previous investigations showed that quantitative immunofluorescence using antigens covalently bound to agarose particles represents a reproducible and sensitive assay for antibodies in experimental antisera. In this paper data are presented which show that also thyroglobulin antibodies in patients sera can be demonstrated using the defined antigen substrate spheres (DASS) system with thyroglobulin as antigen. Sera of 45 patients with various thyroid diseases were investigated for the presence of thyroglobulin antibodies using passive haemagglutination and the quantitative immunofluorescence technique. Comparable results were obtained with both techniques. Advantages and disadvantages of both methods are discussed.

Subclass Typing of IgG Antibodies Formed by Grass Pollen-Allergic Patients during Immunotherapy

The subclasses of IgG antibodies formed by grass pollen-allergic patients during immunotherapy were investigated by using a radioallergosorbent test (RAST) and a quantitative immunofluorescence method known as the defined antigen substrate spheres (DASS) system. By the use of rabbit antisera directed against the subclasses of IgG, the specificity of which was checked in the passive hemagglutination and immunofluorescence techniques, it was shown that a relatively high proportion of the grass pollen-specific antibodies belonged to the IgG4 subclass. Apart from the high binding activity of IgG4 which increased during treatment, a moderate binding activity of the other subclasses was also found. Binding of all subclasses increased slightly in the pollen season and could be specifically blocked by perincubation with soluble grass pollen extract. The results of the IgG4 binding, determined in vitro with the DASS system, and the blocking activity of the sera, determined in vivo by skin tests are suggestive for a relation between these activities. Also in the group of patients with a low IgE-RAST score, the skin reactivity decreased as the IgG4 binding activity increased.

Schistosoma mansoni: Immunohistoperoxidase procedure in Defined Antigen Substrate Spheres ( DASS) system as serologic field test

A promising serological field test for schistosomiasis could be developed by means of an immunohistoperoxidase procedure in the Defined Antigen Substrate Spheres (DASS) system. Sera of individuals with Schistosoma mansoni infections were examined with the DASS-system, using peroxidase labeled conjugates. The results were compared with those obtained with the Indirect Fluorescent Antibody (IFA) technique on frozen sections of the adult parasite. The DASS-system proved to give specific positive reactions at higher serum dilutions than the IFA-technique. The results of the test could readily be observed with the naked eye.

THE DEFINED ANTIGEN SUBSTRATE SPHERES (DASS) SYSTEM AND SOME OF ITS APPLICATIONS*

A quantitative immunofluorescent technique based on the covalent coupling of protein to Sepharose beads, the so-called defined antigen substrate spheres (DASS) system is described, and the main technical aspects are discussed. The method proved to be reproducible and has a lower detection limit equivalent to the amount of antigen, present per bead, that can bind 1 pg fluorescent labeled antibody. The DASS system has many applications, and some of them are discussed, such as the quantitation of antibodies and antigens with Sepharose-coupled antigens or antibodies. Not only antigen-antibody interactions but also any reaction that results in a stable complex can be studied, provided antisera against one of the constituents are available. An example is given by the complement fixation to Sepharose-coupled IgG. For the quantitation of hydrophobic antigens, hydrophobic Sepharise can be used.

An immunofluorescence reaction for Fasciola hepatica using the Defined Antigen Substrate Spheres (DASS) system

The Defined Antigen Substrate Spheres (DASS) system, using Fasciola hepatica antigens, proved to be a promising immunofluorescent antibody test for fascioliasis. The antigen bound beads could be freeze-dried and reconstituted to a spherical form for measurement. Sera of individuals with F. hepatica infections were examined with the Indirect Fluorescent Antibody (IFA) technique on frozen sections of the adult parasite and with the DASS system. Sera of experimentally F. hepatica infected rabbits were examined with the IFA technique, the DASS system, and the Soluble Antigen Fluorescent Antibody (SAFA) technique. A few bovine sera with F. hepatica infection were examined with the DASS system.

A quantitative immunofluorescence method for the determination of IgE antibodies in comparison with the radioallergosorbent test (RAST)

A quantitative immunofluorescence technique, the defined antigen substrate spheres (DASS) system, was tested for its suitability for IgE antibody detection. The method is based on the binding of IgE antibodies to a Sepharose-coupled allergen and subsequent quantitation of the bound IgE by a fluorescent anti-IgE antibody. The sensitivity of the method was compared with that of the radioallergosorbent test (RAST), an equivalent radioactive technique. Although the DASS technique was found to be about 10 times less sensitive than the RAST, its sensitivity warrants clinical use, provided highly purified allergen extracts are applied. The method is also suitable for quantitation of the total IgE content in sera with a lower limit of detection of 10 IU/ml serum.

Immunoprecipitation and class-specific immunofluorescence titration of human serum antibodies to Schistosoma mansoni antigens.

Twenty sera of individuals with Schistosoma mansoni infections were examined by means of immunoelectrophoresis, Indirect Flourescent Antibody (IFA) technique and the Defined Antigen Substrate Spheres (DASS) system. Immunoprecipitins against S. mansoni antigen and against antigen of the intermediate host, Biomphalaria glabrata, were demonstrated. The presence of specific anti-S. mansoni IgA, IgM, IgG and IgE antibodies were demonstrated. The presence of specific anti-S. MANSONI IgA, IgM, IgG and IgE antibodies was identified with the IFA-technique on frozen sections of adult parasites. The DASS-system proved to be more sensitive than the IFA-technique.

Microfluorometry of antigen-antibody interactions in immunofluorescence using the defined antigen substrate spheres (DASS) system. Sensitivity, specificity and variables of the method

Antigens covalently bound to spherical agarose beads were used as an artificial substrate for quantitative immunofluorescence as described previously. The defined antigen substrate spheres system (DASS system) has been further investigated in regard to important variables of the method. The influence of the density of the beads suspension, of the antigen concentration per bead volume and of the incubation time on the resulting fluorescence intensity was investigated. An immunofluorescence staining procedure standardized according to these investigations resulted in a reproducible assay. The sensitivity of the system was tested using conjugates and antisera with defined antibody content. Anti-IgG antibodies could be detected with the direct immunofluorescence technique in a concentration of 100 ng/ml and anti-ovalbumin antibodies with the indirect immunofluorescence technique in a concentration as low as 10 ng/ml. The specificity of the DASS system has also been investigated with classical methods for testing the specificity of immunofluorescence reactions, such as the neutralisation and, the blocking test. It was found that specific reactions could be completely inhibited by absorption with the appropriate antigen and significantly reduced but not completely abolished by blocking with unlabelled antiserum. The possibilities of the DASS system in fundamental and applied immunofluorescence studies are discussed.