Defined Antigen Substrate Spheres System Research Articles

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.

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.

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.

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.