Isolation and partial characterization of the tegumental outer membrane of schistosomula of Schistosoma mansoni

Separation of the external membranes from freshly converted mechanical schistosomula of Schistosoma mansoni was achieved by osmotic shock under hypertonic conditions, followed by mechanical shearing and ultracentrifugation. Prior to treatment, the schistosomula were surface labeled by introduction of N-DNP-epsilon-aminocaproylphosphatidylethanolamine molecules into their lipid bilayer followed by anti-DNP antibodies and stained with either 125I-protein-A or ferritin labeled secondary anti-DNP antibodies. This label provided a membrane marker by which the purity of the preparation could be assessed at each stage. Fluorescence staining with FITC-conjugated secondary antibodies prior to treatment revealed that the homogeneously stained membrane of the intact schistosomula became swollen and ruptured after the osmotic shock. The isolated membrane pellet was intensely fluorescent. Electron microscopical examination revealed mostly vesicles, some of them with organized multilayer assembly. The vesicles were ferritin labeled, indicating that they originated from the outer surface membrane of the schistosomula. A 100 fold enrichment in the alkaline phosphatase activity and about 300 fold enrichment in acetylcholinesterase activity in the membrane preparations, as compared to the intact schistosomula, was found. The isolated tegument was analyzed by SDS-polyacrylamide gel electrophoresis. The pattern obtained showed three major bands, of molecular weights 69 000, 45 000 and 12 000 alongside with a large number of minor bands. Immunoprecipitation of the isolated 125I-labeled membrane antigens with antisera from chronically infected mice revealed these three major bands together with three other bands of molecular weight 38 000, 23 000 and 16 000.     

Interaction of immunoglobulin with actin

Actin can form specific, direct associations with immunoglobulin resulting in soluble complexes or cross-linked matrices. This interaction can be detected by four in vitro assays using purified components: (1) actin enhances the cytophilic activity of guinea pig IgG2; (2) in solutions of low ionic strength, actin and IgG2 co-precipitate: (3) soluble complexes exist in 0.1 M KCl as revealed by the displacement of actin from its expected sedimentation pattern in a gradient of sucrose when in the presence of IgG 1, IgG2, or IgM; (4) immunoglobulin (IgG1, IgG2, BGG)‡: increases the viscosity of F-actin solutions, presumably by crosslinking F-actin filaments. These data suggest that direct interaction of a cytoskeletal protein with a cell surface receptor is possible.     

An improved autoradiographic technique for the detection of antibody-forming cells

An autoradiographic technique for the detection of antibody-forming cells has been developed for the assay of anti-DNP responses. The lymphoid cells suspension to be assayed was allowed to sediment on to a glass slide coated with DNP-conjugated gelatin to which the secreted antibody bound during subsequent incubation. The bound antibody and its Ig class was revealed by a second incubation using ¹²⁵I-anti-immunoglobulin reagents followed by autoradiography. Studies on the sensitivity and specificity of the method are presented and its advantages over other techniques described. The technique should be readily applicable to other haptens.     

Cross reactions to antigens causing hypersensitivity pneumonitis

The sera of patients with asthma, aspergillosis, pigeon breeder's disease, farmer's lung, and a hypersensitivity pneumonitis in textile plant workers were screened by gel diffusion against a variety of fungal antigens and dust extracts. Patients with hypersensitivity pneumonitis were especially prone to develop precipitating antibody to numerous airborne antigens. From the results it was concluded that patients with hypersensitivity pneumonitis form precipitating antibodies to definite groups of antigens in their environment. Most of the patients had precipitins to house dust that formed lines of identity with fungi and thermophilic actinomycetes.     

The separation of human serum IgG into subclass fractions by immunoaffinity chromatography and assessment of specific antibody activity

Murine monoclonal antibodies ( McAbs ) with specificity for subclass-specific or subclass-restricted determinants on human IgG have been coupled to Sepharose to generate affinity columns. The judicial use of positive and negative chromatography and the exploitation of the special properties of individual McAb affinity columns has allowed the preparation of individual IgG subclasses from polyclonal IgG containing less than 1% contamination by any other IgG subclass. The specificity of the antibodies present in each polyclonal IgG subclass preparation has been assayed against a bacterial toxoid (tetanus), 2 bacterial cell wall antigens (E. coli and pneumococcal) and coat antigen(s) of a DNA virus (CMV). Antibodies were predominantly IgG1 to tetanus toxoid, IgG2 to pneumovax and E. coli cell walls, and IgG1, 2 and 3 to CMV coat antigens.     

TNP-enzyme conjugates for the detection of anti-TNP antibody producing cells in vivo

After injection of TNP-KLH in mice and TNP-BGG-PEN in rabbits, anti-TNP antibody-forming cells were observed in the spleen. When cryostat sections of the stimulated spleen were incubated with TNP-HRP conjugate, and then treated for HRP cytochemistry, the cytoplasm of anti-TNP-forming cells was stained red. When similar sections were incubated with TNP-AP conjugate, and then treated for AP cytochemistry, the cytoplasm of anti-TNP-forming cells was stained blue. After simultaneous incubation with TNP-AP conjugate and PEN-HSA-HRP conjugate, followed by treatment for HRP cytochemistry and AP cytochemistry, anti-TNP-forming cells with a blue cytoplasm and anti-PEN-forming cells with a red cytoplasm could be distinguished in the same spleen section.     

Comparison of methyl-p-hydroxybenzimidate and 1,3,5-trichlorotriazine in producing sensitive target cells for use in the hemolytic spot assay

Two bifunctional reagents were shown to be useful in the coupling of immunogens to the surface of either nucleated or non-nucleated cells. The heterobifunctional reagent methyl-p-hydroxybenzimidate was used to couple aromatic amino-haptens to the surface of SRBC which resulted in a stable and sensitive target cell capable of detecting as little as 20 pg of purified anti-hapten antibody in the hemolytic spot assay. The multifunctional reagent 1,3,5-trichlorotriazine yielded similar results when coupling amino-haptens to the surface of SRBC for use in the hemolytic spot assay. This reagent was also used to couple protein to the surface of SRBC which were able to detect as low as 1 ng of purified anti-protein antibody in the hemolytic spot assay. The sensitized SRBC produced using either of these coupling reagents were shown to remain stable for several months giving reproducible results from one test to another. Lastly, 1,3,5-trichlorotriazine was used to couple the hapten 4-aminophthalate to the surface of nucleated cells with retention of >90% cell viability with continued growth and cellular division in culture.     

Effect of reduction and alkylation on structure and function of rabbit IgG antibody—I. Effect on ability to activate complement depends on conditions of reduction

Anaerobic reduction of rabbit IgG antibody with either dithiothreitol at pH 8.0 or 2-mercaptoethanol at pH 5.0 results in the production of antibody molecules which lack haemolytic activity yet whose interchain disulfide bonds remain intact. Aerobic reduction with 2-mercaptoethanol results in more haemolytic activity than can be accounted for by the residual amount of Ab with intact interchain bonds. Taken together, these findings suggest that the interheavy chain disulfide bond in rabbit IgG is not essential for interaction with complement but that an intraheavy chain bond is critical.