L. Tučková

Lytic activities in coelomic fluid of Eisenia foetida and Lumbricus terrestris

Coelomic fluids of the two earthworm species E.foetida (E.F.) and L.terrestris (L.T.) have not only the ability to lyse various vertebrate erythrocytes but also to digest vertebrate serum proteins. Both activities are carried by different molecules since hemolysis but not proteolysis was inhibited by simple sugars. In contrary, proteolysis was blocked by PMSF which did not influence hemolysis. Coelomic fluids of E.F. digest effectively vertebrate serum proteins (PIgG, HSA) but not the proteins of L.T. coelomic fluids. The proteolytic activity was detected in approximately 40 000 mol. wt. fraction. After digestion proteolytic fragments were analyzed by immunoelectrophoresis, SDS-PAGE and TCA precipitation. Two of the fragments reacting with PIgG antisera remained intact even after 120 h digestion.

The fate of protein antigen in earthworms : study in vitro

Earthworm coelomocytes digest protein antigen in vitro. Proteolytic activity was detected both in cell-free coelomic fluid and in cell cultures of free coelomocytes which are effectors of earthworm immunodefense mechanisms. Antigen is cleaved either intracellularly or by proteolytic enzymes released by coelomocytes into the medium. Proteolysis was observed both in non-stimulated and antigen-stimulated cultures. Since significantly higher proteolysis was shown in supernatants from cultures of antigen-stimulated coelomocytes, we can assume that the release of proteolytic enzymes was inducible.

Receptor-specific fractionation of immunocompetent cells and purification of antibodies on hydroxyalkyl methacrylate immunosorbents.

Abstract Immunosorbents prepared with hydroxyalkyl methacrylate gels activated by cyanogen bromide, have a binding capacity for specific antibodies from immune sera similar to that of commercial Sepharose immunosorbents. Moreover, they can be used in specific fractionation of immunocompetent cells. Enriched cells suspensions of antibody producing spleen cells from rabbits immunized with SRBC or HSA were obtained by using large gel particles (500–700 μm). Using the batch method, the antibody producing cells could be concentrated 5–30 times. Blocking of cell receptors with anti-immunoglobulin serum or specific antigen confirmed the specificity of cell attachment to the immunosorbent. According to the results obtained hydroxyalkyl methacrylate gels are suitable for concentration of memory cells and separation of T and B cells.

Monoclonal antibodies to antigen binding protein of annelids (Lumbricus terrestris)

Abstract 1. 1. Monoclonal antibodies (mAb) to antigen binding protein (ABP) of the earthworm Lumbricus terrestris have been prepared. 2. 2. The specificity of mAb for a determinant located outside the antigen binding site was determined and verified in inhibition experiments. 3. 3. The mAb were used for isolation of a 56 kDa ABP by an immunoprecipitation technique. 4. 4. The binding of mAb to coelomocytes has demonstrated the existence of two cell populations, one with low and the other with high densities of ABP molecules on the cell membranes.

Isolation and properties of antigenic-specific receptors from rabbit and pig lymphoid cells

Abstract In order to study the character and properties of antigen-specific receptors on lymphocyte surfaces, an immunoadsorption method based on the principle described by Kiefer (1973, 1975) has been developed. This method permits isolation of receptors from rabbit and pig spleen and lymph node cells in quantities sufficient for basic analyses. Isolated receptors represent a mixture of two groups of antigen binding molecules. Both groups react with corresponding anti-idiotypic antisera but only one of them reacts with antisera to serum immunoglobulins. These two fractions can be separated using an immunoadsorbent with bound antibodies to serum immunoglobulins. In the fraction bearing immunoglobulin determinants, the presence of IgM and IgG but not IgA has been detected. The IgG receptors and IgG antibodies in the serum of the same animal had different isoelectrofocusing spectra. The origin of the other antigen-binding fraction remains to be established. The results obtained with antisera to the ‘constant part’ of this molecule confirmed that it does not carry conventional immunoglobulin determinants.

Cross-reactivity of human and a putative pig IgD. Pig IgD-like molecules as serum and lymphocyte components.

Proteins of pig serum and splenocytes which were isolated on immobilized guinea pig antibodies against human IgD were characterized by SDS-PAGE. In both cases the main isolated components possessed several properties nearly identical with those of human IgD: molar mass of the components and their chains, the ratio of radioactivity of their heavy and light chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains in the native molecule to trypsin digestion. These IgD-like components not adsorbed on immobilized normal guinea-pig IgG. We suggest that the components of pig serum and splenocytes cross-reacting with highly specific antibodies against human IgD represent candidates for pig IgD. The percentage of the IgD-like molecules on the surface of pig splenocytes labeled with the antibody against human IgD was similar to the percentage of splenocytes bearing all immunoglobulins. Therefore, we suggest that IgD-like molecules were occurring on the surface of cells mostly together with molecules of another immunoglobulin class.

Influence of proteolysis inhibitors and immunoadsorption technique on the composition of rabbit antigen-specific receptor preparations. Presence of Fc receptors

Addition of proteolysis inhibitors during the isolation procedure of rabbit arsanylated bovine IgG specific receptors decreased significantly the amount of low-molar-mass proteins in all receptor preparations.Rabbit ARS-BGG-specific receptor preparations isolated by immunoadsorption technique contain molecules which do not react with antigen and antibodies against immunoglobulins and have identical molar mass andchymotryptic peptide composition as those of isolated Fc receptors. It is suggested that during isolation of antigen-specific receptors from the surface of lymphoid cells, Fc receptors react with complexes composed of antigen and Ig+ receptors on the surface of immunoadsorbent and are isolated together with antigen-specific receptors.

Characterization of antigen-specific receptors from pig lymphocytes

Antigen-specific receptors were isolated from the surface of spleen and lymph node cells of ARS-BGG-stimulated miniature pigs using an immunoadsorption technique. The yields of the receptors can be increased by 20-hr cultivation of the cells protein-free medium prior to the isolation procedure. In this case antigen-specific molecules can be isolated not only from the surface of the cells but also from the culture medium. 125I-labelled isolated receptors were separated by SDS-PAGE into six fractions. The first corresponded to aggregated molecules and IgG, the mol. wts of the other fractions were 56,000, 35,000, 26,000 and 10,000. It has been shown that they all were able to bind antigen but only the first two reacted with anti-Ig antibodies and Staphylococcus aureus protein A-Sepharose 4B. Gel filtration of the same sample of 125I receptors on Sephacryl S-200 revealed the presence of four peaks. Three of them were able to bind antigen as detected by radioimmunoassay. Fractions of all three peaks reacted also with anti-Id and anti-R (anti-Ig- receptors) antisera, but only fractions of the first two peaks reacted with anti-Ig antisera. The finding that Ig- receptors and more heterogeneous than Ig+ receptors is discussed. No reaction of isolated 125I receptors with anti-SLA and anti-SLB antisera used was detected in the solid-phase radioimmunoassay.

The interaction of staphylococcal protein a with free coelomocytes of annelids

The Staphylococcal protein A (SpA) binding protein was detected on the surface of annelid coelomocytes. The flow cytometric analysis revealed that 50% coelomocytes of Lumbricus terrestris react with SpA, a figure six times higher than the number of positive coelomocytes found in Eisenia foetida.

Role of saccharides in immunoglobulin--Fc receptor interactions.

The rosettes formed by mouse peritoneal macropahges or DCH-5 cells and TNP-erythrocytes coated with anti-TNP antibodies of different isotypes were inhibited to various extent by monosaccharides. The most effective inhibitors were N-acetylglucosamine, glucosamine, mannose and N-acetylneuraminic acid in 1–5 mmol/L concentrations. Even more efficient were glycopeptides isolated from IgG molecules. The Fc receptors (FcRs) released from DCH-5 cells during cultivation and gradually separated by affinity chromatography on immobilized IgG reacted with aggregated IgG and inhibited the rosette formation. The FcRs eluted by monosaccharides influenced mainly the number of rosettes mediated by IgA and IgE while those eluted with a glycine-HCl buffer inhibited preferentially IgG rosettes. As shown by SDS-PAGE the heterogeneity of the fraction eluted with a mixture of monosaccharides revealed one main component with an effective molar mass of 50 kg/mol. The glycine-HCl eluate contained two major components of 55 and 38 kg/mol. The IgG-Sepharose 4B bound all the fractions but only the binding of the 50 kg/mol molecule could be inhibited by monosaccharides.