Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

Detection of radiolabelled bone marrow cells bearing surface ace immunoglobulins by combined autoradiography and immunoperoxidase

A method is described for the simultaneous detection of radiolabelled bone marrow cells bearing surface immunoglobulins by combined autoradiography and immunoperoxidase. Bone marrow cells from normal CBA mice prelabelled in vivo with ¹²⁵IUDR or exposed in vitro to [³H]thymidine were incubated with rabbit anti-mouse immunoglobulins under capping conditions, washed, cytocentrifuged and treated with methanol and hydrogen peroxide to destroy endogenous peroxidase. Cells were then covered with peroxidase-conjugated goat anti-rabbit immunoglobulins, washed, treated with diaminobenzidine a and hydrogen peroxide and finally covered with autoradiographic stripping film and exposed for different times. Peroxidase-positive cells were typically capped and those radiolabelled had autoradiographic silver grains overlying the nucleus.     

A method for the accurate determination of anti-hapten cytotoxic T lymphocyte precursors: correction for apparent 'anti-self' reactivity

A method is described for accurately determining the frequency of precursors of hapten specific cytotoxic T cells. The method is based on a standard Poisson analysis of limit dilution cultures, but makes a correction of 'anti-self' reacting clones and for spontaneously arising clones that recognise modified self. These corrections are shown to be especially important when low hapten densities are used, where there may be more than a 10-fold difference between the corrected and uncorrected frequency estimates. Determined levels of antigen specificity and of H-2 restriction are significantly enhanced by application of this method.     

Acquired α2 macroglobulin on the surface of human lymphoblastoid cells

The molecular nature of the membrane antigen that is acquired from FCS-containing media by human lymphoblastoid cells has been investigated. The presence of bovine α₂, macroglobulin on the surface of Namalva cells was demonstrated by radioimmunoassay using specific antisera. Alternatively, cell-bound bovine α₂,M could be detected by the more sensitive heterophile rosette assay described previously. Namalva cells grown in NHS-containing media acquired bovine α₂ M upon subsequent incubation with the purified protein in a dose- and time-dependent way. Acquisition of α₂ M was demonstrated using both viable and formaldehyde-fixed cells. Purified fetuin, which carries a heterophile epitope shared with bovine α₂ M as well as with other glycoproteins, failed to bind with the membrane of Namalva cells. The possible role of acquired α₂ macroglobulin on cells has been discussed.     

Technical improvements in the clotted plasma droplet MIF assay in vitro.

The migration inhibitory activity of culture supernatants of rat and human lymphocytes stimulated with PHA and Con A-Sepharose was tested on cell migration from clotted plasma droplets. This technique was improved by using homologous as well as heterologous plasma and fibrinogen solutions for suspending the migratory cells, automatic micropipettes for performing the technique and purified cell populations as target. The effects of calcium chloride and of the cell concentration in the plasma droplets on the migration indices obtained in the MIF assay were tested.     

Ability of rosetting or non-rosetting individual control and inflammatory macrophages to kill Escherichia coli X43 intracellularly

An autoradiographic method combined with a rosette technique was used to assess the bactericidal activity of individual control and inflammatory peritoneal macrophages (PM phi) in the presence or absence of expression of Fc receptor for IgG (FcR). There was a lack of FcR reactivity in a certain percentage of both categories of PM phi exposed to E. coli X43, a bacterium which is readily phagocytosed in the presence of specific antibody. Both rosetting and non-rosetting PM phi were capable of phagocytosing E. coli X43, but inflammatory PM phi showed a marked reduction in their capacity to ingest these bacteria compared with control PM phi. Once ingested the E. coli X43 were killed equally well by non-rosetting and rosetting control and inflammatory PM phi.     

Chemical Characterization of Macrophage Cytotoxicity Factor,Macrophage Migration Inhibitory Factor,T-Helper Cell-Replacing Factor and Colony-Stimulating Factor from Culture Supernatants of Concanavalin A-Stimulated Murine Spleen Cells

Supernatants from Concanavalin A-stimulated murine spleen cells were subjected to hydrophobic interaction chromatography on phenyl-Sepharose. Macrophage cytotoxicity factor (MCF), macrophage migration inhibitory factor (MIF), T-helper cell-replacing factor (TRF) and colony-stimulating factor (CSF) were bound at high ionic strength and were released stepwise at low ionic strength. CSF thus could be separated from MCF, MIF and TRF and the bulk of other proteins. Chromatography of pools containing MCF, MIF and TRF on Sephadex did not lead to a separation of the three activities which were all found in a molecular weight range of 25.000-55.000. Isoelectric focusing of these pools in pH range from 4 to 9 gave two peaks for MCF at pH 8.2 and 7.2, whereas MIF activity focused from pH 4.5 to 5.5. TRF activity was found in a single sharp peak at pH 5.3. The results demonstrate that the four biological activities can be distinguished on a chemical basis and are accessible for purification and chemical characterization.     

An enzyme-linked immunosorbent assay (ELISA) for detection of Fc alpha receptors on isotype-specific T cells

A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) has been developed using T cell hybridomas as coating antigen, for detection of Fc receptors for IgA (Fc alpha R). T-T hybridomas were generated from fusions of Fc alpha R+ T cell clones from mouse Peyer's patches with the Fc alpha R- R1.1 T lymphoma cell line. The 2 T-T hybridomas (designated Th HA) used here express Fc alpha R as determined by a rosette method and by ELISA. Th HA cells were cultured under conditions for maximum Fc alpha R expression, were added to individual wells of 96-well EIA plates, and were fixed in situ with glutaraldehyde. Plates were incubated with purified mouse monoclonal IgA, IgM or IgG1 and were developed with beta-galactosidase-coupled goat IgG antibodies specific for mouse heavy chains. Using the ELISA, both Th HA cell lines were shown to express significant levels of Fc alpha R, lower but detectable Fc mu R, and no discernible Fc gamma 1R. Interestingly, the rosette assay only allowed detection of receptors for IgA. When splenic lymphocytes were used, good Fc mu R and less Fc alpha R expression occurred on these cells as determined by ELISA and rosetting; however, no Fc gamma 1R cells were detected by either method. Thus, the ELISA is sensitive and reproducible, and allows an objective measurement of FcR expressed on T cells.     

Determination of oxidative stress parameters and DNA fragmentation on post-thawed buck semen in the presence of ram seminal plasma and fetal calf serum

The aim of the study was to investigate the efficiency of ram seminal plasma and fetal calf serum on freezing of buck semen. Twenty ejaculates were collected using an electro-ejaculator and split into six groups. While FCS additive was not used in A1, A2 and A3 groups, 10% FCS was added to B1, B2 and B3 groups. These groups were then edited according to whether the buck or ram SP was involved. The design of the groups was done as follows: Group A1 (control 1), group A2 without buck SP, group A3 containing ram SP instead of buck SP. Groups B1 (control 2), B2 and B3 were the FCS added forms of these groups. Progressive sperm motility percentages in Group A1 and Group B2 were found to be higher when compared to the lowest Group B3. There were no significant differences between the groups in neither the levels of reactive oxygen species nor the enzyme and glutathione activities. In conclusion, the lack of statistical difference between the groups suggested that despite the supplements used but only when the buck spermatozoa structure was healthy, the cell could preserve acrosome, DNA and the integrity of membrane.     

Human Peripheral Eosinophils with Receptors for IgM: Demonstration and Ultrastructural Morphology

Receptors for IgM were detected on peripheral blood human eosinophils by a rosette technique with ox red blood cells coated with the IgM fraction of the specific immunserum. Between 14 % and 43 % (mean 27 %) FcµR positive cells were found after an overnight incubation period at 37°C by using this technique. The specificity of the receptors for IgM was assessed by studying the inhibitory capacity of purified human IgM in the rosette assay. From an ultrastructural point of view, the EA^M rosette-forming cells are mature eosinophlic granulocytes characterized by a nucleus with a variable number of lobes and a certain number of «first type» granules partially or totally devoid of their content.