Comparison of the immune response to Ars-BGG in germfree or conventional piglets.

Neonatal germfree (GF) colostrum-deprived and conventional (CV) colostrum-fed piglets were immunized IP with p-azo-phenyl-arsonate-bovine gamma globulin (Ars-BGG) in Freund's adjuvant to study the development of the immune response in the absence or presence of maternal antibodies and environmental antigens. Overall, the immune response varied greatly within each group but did not differ in GF from CV piglets statistically. Affinity immunoblot analysis suggested that anti-Ars antibody was more restricted in GF than CV piglets and clonotype shifts occurred more in GF than CV piglets after each antigenic stimulation. In contrast, the clonotype pattern of the anti-BGG antibody was similarly heterogeneous in the two groups. Based on the affinity immunoblot data the antibodies generated to the Ars-haptenic group in CV piglets are more heterogeneous than GF piglets and suggest that clonotype generation is influenced by maternal antibodies and environmental antigens.

Compartmentalized reactivity of M3/38 (anti-Mac-2) and M3/84 (anti-Mac-3) in the uterus of pregnant mice.

Changes in the expression of antigens on mouse uterine or embryonic tissues were examined by enzyme immunocytochemistry. Cryostat sections of uteri from days 1, 8 and 15 of pregnancy were probed with the monoclonal antibodies M3/38 and M3/84, originally defined by their reactivity with macrophage surface antigens (Mac-2 and Mac-3, respectively). In the uterus of pregnant mice, reaction of these antibodies was not limited to leucocytes. M3/38 did not react at detectable levels with cells in the uterus on day 1 but did react with decidual cells immediately surrounding the embryo on day 8. By day 15, the placenta, decidua basalis and metrial gland were intensely positive but the embryo was negative. M3/84 reacted with the luminal side of the endometrium on day 1, the entire decidual mass on day 8, and with all maternal and fetal tissues on day 15. M3/38 was detected in saline-soluble preparations of uterine tissue and had a molecular mass of approximately 32-35 kDa as determined by SDS-PAGE and western blot analysis. The pattern of expression of these molecules suggests a functional relationship to developmental changes that occur at the maternal-fetal interface.

Changes in surface antigens on preimplantation mouse embryos.

Changes in the expression of specific cell surface antigens on preimplantation mouse embryos were examined by immunocytochemistry. Embryos were recovered at various times during the preimplantation phase of normal pregnancy, and from pregnancies with experimentally induced delayed implantation, and were probed with a panel of monoclonal antibodies against murine leukocyte antigens. Antibodies directed against certain macrophage surface glycoproteins (i.e., Mac-2 and Mac-3) and those against lysosome-associated membrane glycoproteins (i.e., LAMP-1 and LAMP-2) reacted specifically with cell surface determinants on the embryos. Differences in the spatiotemporal patterns of antibody binding during normal and delayed implantation indicate that expression of the antigenic determinants recognized by these antibodies is regulated individually in response to intrinsic as well as extrinsic signals at the time of implantation, and thus they may be important for the process of embryo implantation.

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation.

A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.

Comparative studies of recycling isoelectric focusing and continuous flow electrophoresis: separation of proteins with minor charge differences.

Continuous flow zone electrophoresis (CFE) and recycling isoelectric focusing (RIEF) are two of the alternative formats for fluid phase preparative isolation of biological products in liquid separation media. The McDonnell Douglas CFE system has been used for both ground-based and microgravity separations. The ground-based McDonnell Douglas CFE and RIEF were compared for the ability to resolve mixtures of proteins with known charge differences. Mixtures of 1) cytochrome c, myoglobin, and ovalbumin or 2) beta-lactoglobulin and ovalbumin were used to evaluate the resolving capabilities of CFE and RIEF. Following separation, fractions were analyzed by determining absorbance at 280 nm and by analytical isoelectric focusing (IEF) using Coomassie Brilliant Blue or silver staining to detect focused proteins. Both CFE and RIEF apparently separated the components of both mixtures into individual peaks, separated by fractions which contained little or no detectable protein. Coomassie-stained analytical IEF gels supported this finding. However, when separated proteins were analyzed by silver staining of the analytical gels, the separation of ovalbumin from beta-lactoglobulin by CFE was not complete. Ovalbumin was free of beta-lactoglobulin but beta-lactoglobulin was contaminated by trace amounts of ovalbumin. RIEF clearly separated each protein with no detectable contamination. These data demonstrate the superiority of RIEF over CFE for resolution of protein mixtures having only minor charge differences. RIEF may be more efficient due to the documented electrodissociation of noncovalent protein:protein complexes which occurs during RIEF separations.

Isoelectric focusing analysis of antibody clonotype changes occurring during immune responses using immobilized pH gradients.

Serum was collected from rabbits at 2-day intervals following a single injection with tetanus toxoid or at weekly intervals following multiple injections with Micrococcus lysodeikticus cell walls. These sera were analyzed for the presence of individual clonotypes of specific anti-tetanus or anti-micrococcal antibodies by isoelectric focusing in immobilized pH gradients with added carrier ampholytes followed by affinity immunoblotting. The affinity immunoblots obtained clearly defined both the rapid disappearance and late appearance of distinct subsets of antibody clonotypes during the response. These data demonstrate the application of affinity immunoblotting combined with immobilized pH gradients for detecting the subtle changes in specific antibody clonotype patterns which occur during an immune response.

Production and assay of ovine T cell growth factor by concanavalin A stimulated peripheral blood leukocytes.

Conditions for the production and assay of ovine T cell growth factor (TCGF) activity were evaluated. Peripheral blood leukocytes were stimulated with concanavalin A (Con A) in the presence of 2% autologous serum or serum-free media. Supernatants were harvested after 30 hr and concentrated for further characterization. A 28 hr proliferation assay with 2.5 X 10(4) 24 hr Con A blasts per well was optimal for detection of TCGF. Peak TCGF activity occurred with a 30-37 KD molecular weight fraction. Production and assay of TCGF were performed under autologous conditions to reduce background stimulation which occurred when fetal bovine serum was present. This methodology required no cell lines or inbred animals and should be adaptable to the study of immunostimulatory molecules of other outbred species.

Affinity immunoblotting. High resolution isoelectric focusing analysis of antibody clonotype distribution.

A sensitive and specific method has been developed for analyzing specific antibody clonotype changes during an immune response or comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies were separated by flatbed acrylamide isoelectric focusing (IEF) and analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels after IEF bound the antigen on the nitrocellulose when the coated nitrocellulose was laid over the gels. Non-specific protein binding was inhibited with Tween 20. Bound IgG antibody clonotypes were detected using peroxidase-conjugated anti-IgG. This method has been used for the analysis of Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentration for coating the nitrocellulose membranes ranged from 10 to 100 micrograms/ml. The reactions could be inhibited by saturating the nitrocellulose with soluble protein antigen or free hapten prior to immunoblotting. Antibodies of alternative allotypic forms were detected by probing the immunoblot with biotinylated anti-allotype antibodies instead of anti-IgG. The simultaneous analysis of alternative allelic forms of antibodies of defined specificity is not possible with methods which use labelled antigen for clonotype detection.

Fluorescein isothiocyanate as a cell marker for tracing lymphocyte circulation in sheep.

The use of fluorescein isothiocyanate (FITC) as a cell marker in ovine lymphocyte circulation studies was investigated. The effects of the label on lymphocyte function were assessed by lymphocyte blastogenesis, cell-mediated cytotoxicity and normal lymphocyte transfer reactions. Labelling lymphocytes in 0.05 mg FITC/ml for 10 min at 37 degrees C was found to be optimum. Circulation kinetics of FITC-labelled efferent lymph lymphocytes were monitored as they disappeared from peripheral blood following intravenous infusion and reappeared in the efferent lymphatic duct of the popliteal lymph node. No functional changes were detected in lymphocytes labelled with the optimal regimen.