Guinea Pig IgG Research Articles

Leishmania enriettii amastigotes: demonstration of fibrillar surface.

An extensive fibrillar surface was identified in situ by transmission electron microscopy on the amastigote stage of the macrophage intracellular parasite, Leishmania enriettii. Attachment to the amastigote surface of 20-160-nm fibrils was clearly evident on organisms that had been isolated from guinea pigs. Surface fibrils were not detected after in vitro transformation of the amastigote to the promastigote. Incubation of amastigotes in heat-inactivated guinea pig serum containing a high titer of antibodies to L. enriettii followed by exposure to ferritin-conjugated antibody to guinea pig IgG resulted in labeling of surface fibrils, a result indicating that these fibrils are of parasite origin. Shedding of labeled fibrils was also evident.

Binding and degradation of soluble immunoglobulin aggregates by mouse mononuclear phagocytes—Stimulation by colony-stimulating factor

The binding and degradation of soluble guinea pig IgG 2 aggregates by murine mononuclear phagocytes were studied. Bone marrow mononuclear phagocytes cultured in the presence of an embryonic fibroblast-conditioned medium (CM) degraded the aggregates to a much greater degree than did resident peritoneal macrophages. Binding and degradation by resident peritoneal macrophages were enhanced by culture in the presence of CM. Freshly harvested thioglycollate-induced peritoneal macrophages bound and degraded the aggregates to the same degree as the cultured bone marrow mononuclear phagocytes did. However, the thioglycollate-induced macrophages lost most of these capacities when cultured in vitro without CM. When CM was added to these cultures, the capacity to bind and degrade was restored in a dose-dependent fashion. To obtain the maximum effect, exposure to CM must be maintained for more than 2 days. The effect of CM could be reproduced with purified CSF-1. Taken together the results of this study indicate that Fc receptor expression is modulated by CSF-1.

Separation of guinea pig IgG subclasses by affinity chromatography on protein A-Sepharose

Guinea pig serum was chromatographed on a column of protein A-Sepharose equilibrated in citrate-phosphate buffer at pH 7.3. The bulk of serum proteins eluted in the starting buffer. Two peaks of protein A-bound serum proteins were eluted by a decreasing pH gradient, a smaller peak centered at pH 4.7 and a larger peak centered at pH 4.3. IgG contained in the peak eluted at pH 4.7 had fast γ immunoelectrophoretic mobility and IgG in the peak eluted at pH 4.3 had slow γ mobility. Antiserum to guinea pig IgG, when absorbed with the pH 4.7 peak, reacted only with the pH 4.3 peak. Antiserum to guinea pig IgG 1, when absorbed with the pH 4.3 peak, reacted only with the pH 4.7 peak. The IgG in the pH 4.7 peak had the immunoelectrophoretic and antigenic characteristics of IgG 1 and the IgG in the pH 4.3 peak had the characteristics of IgG 2. The two subclasses were efficiently separated by pH-dependent affinity chromatography on protein A-Sepharose. The IgG 1 in the pH 4.7 peak was contaminated with 10.8% IgG 2, and the IgG 2 in the pH 4.3 peak was contaminated with 2.7% IgG 1.

Effects of various protease inhibitors on phagocytosis of antigen-antibody complex by macrophages.

In order to characterize proteases of macrophages involved in phagocytosis, the effects of various protease inhibitors were investigated in measuring the uptake and digestion of Bacillus subtilis alpha-amylase complex of guinea pig IgG2 antibody by homologous peritoneal macrophages. The inhibitory effects were observed with diisopropyl fluorophosphate (DFP), L-1-p-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), 1-chloro-3-tosylamido-7-amino-L-2-heptanone (TLCK), antipain, chymostatin, elastatinal and leupeptins, but not with pepstatins. DFP and TPCK reduced both the amounts of antigen-antibody (Ag-Ab) complex taken up by macrophages and of digested products released from the cells. TLCK, antipain, chymostatin, elastatinal and leupeptins also reduced the amount of digested products, but increased that of Ag-Ab complex associated with macrophages. These results suggest that the inhibitory mechanism of DFP and TPCK toward the digestion of soluble Ag-Ab complex by macrophages differs from those of other inhibitors examined.

ISOLATION AND CHARACTERIZATION OF Fcγ RECEPTORS FROM HUMAN PLACENTA

Placental extract was prepared with Tris-HCl buffer pH 7.4 containing EDTA and 2-mercaptoethanol. It agglutinated erythrocytes (E) sensitized with subagglutinating amounts of IgG antibodies from rabbits, guinea pigs and mice, but not E sensitized with IgG from chickens. E or E sensitized with F(ab)2 or IgM antibodies were not agglutinated. The agglutination of EA by the extract was inhibited by human, rabbit and guinea pig IgG, but not by bovine and porcine IgG. Aggregated IgG was more inhibitory than monomeric IgG. IgG3 was the most effective subclass. The extract inhibited the formation of EA rosettes with human mononuclear cells, but did not influence the formation of E or EAC rosettes. The significance of the disulfide bonds and the C gamma 3 and C gamma 2 regions was implied by the finding that the extract neither agglutinated E sensitized with preparations of mildly reduced and alkylated IgG, nor with Facb fragments. These preparations did not inhibit the agglutination. The results strongly indicated that the active component was solubilized placental Fc receptor (FcR). Functionally active FcR was purified by affinity chromatography using aggregated human IgG coupled to Sepharose 4B. SDS-PAGE if the purified FcR under reducing conditions showed one distance band corresponding to approx. 47,000 daltons. The band neither consisted o Ig fragments nor Clq. A rabbit antiserum against the FcR inhibited the agglutination of EA by the extract and stained th FcR-positive areas in placenta.

Distribution of FCγ receptors on isolated gut enterocytes of the suckling rat

An erythrocyte-antibody rosette assay has been used to study the presence and distribution of Fcγ receptors on enterocytes isolated from 12-day-old suckling rat gut by means of a buffer medium containing EGTA. Such receptors were found to be restricted to enterocytes in the proximal region (duodenum and jejunum) of the small intestine and to be acid-pH dependent. For the majority of enterocytes indicator red cells bound in high density to the abluminal plasmalemma but not to the apical microvillous brush border. Since immunofluorescence studies revealed strong binding of added IgG to the microvillous region, a likely explanation is that there is a paucity of Fcγ receptors from the tips of microvilli (at least under the conditions of the rosette assay) and that receptors more deeply situated as inaccessible to indicator red cells was readily inhibited by rabbit, human, guinea pig and rat IgG but less so by bovine IgG, and of the two sub-classes, bovine IgG2 inhibited much more readily than bovine IgG1. Cortisone acetate injection virtually abolished Fcγ receptor expression on isolated enterocytes within three days. These findings correlate both with selective transport of IgG of different species in vivo and the known effect of cortisone acetate to terminate such transport.

Quantitation of specific IgG antibodies in rabbits by a solid-phase radioimmunoassay with 125I-protein A from Staphylococcus aureus.

Radioiodinated protein A (SpA) from Staphylococcus aureus was used to quantitate specific antibodies of the IgG class at the nanogram level in rabbit serum by a standardized radioimmunoassay with the antigen covalently bound to paper discs. Quantitation of IgG in micrograms of IgG per millilitre of serum was done by using a standard curve with purified anti-bovine serum albumin (BSA) IgG or by means of the calculated molar SpA to rabbit IgG ratio of 1:1, thus converting counts per minute of 125I-SpA to nanograms of IgG. The molar ratio for SpA to human IgG was also 1:1. The unspecific binding of IgG to the paper discs was studied and could be depressed by hen serum or by adsorption of the serum sample to cellulose before the assay. The affinity constants of the extracellular SpA and human, rabbit and guinea-pig IgG, respectively, were determined and found to be close to the values previously calculated for cell-wall-bound SpA. The applicability of the assay was demonstrated by quantitation of the specific IgG response on different days after immunization of rabbits with BSA and 3,4-dinitrobenzene (DNP)-BSA.

Effect of pH on the binding between guinea pig IgG isotypes and protein A: evidence for intra-isotypic binding heterogeneity.

Effect of pH on the binding between guinea pig IgG isotypes and protein A: evidence for intra-isotypic binding heterogeneity.

Different functions for specific guinea pig IgG1 and IgG2 in the lysis of sheep erythrocytes by C4-deficient guinea pig serum.

Different functions for specific guinea pig IgG1 and IgG2 in the lysis of sheep erythrocytes by C4-deficient guinea pig serum.

Different affinities of MONO-, DI- and TRI-DNP-ribonuclease a for anti-DNP antibody

2,4-Dinitrophenylated ribonuclease A (DNP-RNase) with 2.6 DNP residues per molecule of RNase was fractionated into DNP 1-, DNP 2-, DNP 3- and more polyvalent RNase by isoelectrofocusing. The measurement of fluorescence quenching revealed that the affinity of DNP 1-RNase for guinea pig IgG2 anti-DNP antibody or its Fab' fragment was markedly lower than those of DNP 2- and DNP 3-RNase. The reduction and alkylation increased the affinity of DNP 1-RNase to those of other polyvalent conjugates, indicating that the single DNP residue of DNP 1-RNase is not easily accessible to active sites of anti-DNP antibody, owing to certain steric hindrance by the tertiary structure of RNase molecule surrounding the DNP-substituted lysine residue. The ability of these conjugates to form complexes with anti-DNP antibody increased markedly with an increase in number of DNP residues substituted to a RNase molecule when estimated by the precipitation with 50% saturated ammonium sulfate; the amount of antigen-antibody complexes was found to be profoundly influenced not only by the affinity of each DNP residue for anti-DNP antibody, but also by the number of DNP residues per molecule of RNase.

Detection of antibodies against foot-and-mouth disease virus using purified staphylococcus A protein conjugated with alkaline phosphatase

Purified protein A (PA) from Staphylococcus aureus was conjugated with the enzyme alkaline phosphatase. This reagent reacted well with pig, rabbit and guinea pig IgG and less well with mouse and bovine IgG. Radioactive 125I-labelled PA showed a similar affinity for the IgGs examined. Antibodies against foot-and-mouth disease virus contained in guinea pig, rabbit and pig serum were detected, using the enzyme-conjugated PA in indirect tests, which were of similar sensitivity to those using enzyme-conjugated anti-species antibody as tracer.

Macrophage handling of soluble immune complexes. Ingestion and digestion of surface-bound complexes at 4, 20 and 37 degrees C.

Radioassays have been developed to measure, as separate events, the ingestion and degradation of macrophage-bound guinea pig IgG anti-DNP (2,4-dinitrophenyl)-DNP-BSA (bovine serum albumin) complexes of defined size and subclass. Complex ingestion was observed to be temperature-dependent and was effectively blocked only by a combination of inhibitors of respiration and glycolysis indicating that the process is under the same metabolic control as fluid phase pinocytosis. On the other hand, the half-life of membrane-bound complexes at 37 degrees C (t1/2 = 5.6 +/- 1.7 min) was considerably shorter than the value expected from membrane turnover studies (t1/2 = 22.4 min) suggesting that complexes are selectively cleared from the cell surface. The rate of ingestion at 20 degrees C was independent of complex size and of the IgG subclass used in complex formation, but was affected by in vivo stimulation of the macrophages before assay. Complex digestion was shown to be highly temperature-dependent and, at 37 degrees C, proceeded at a rate (t1/2 = 15.5 h) which was 20-60-fold slower than the rate of ingestion indicating that the latter is unlikely to influence digestion kinetics. On the other hand, the selective action of 2-deoxyglucose in blocking digestion, but not ingestion, suggests that pinosome-lysosome fusion may play a part in determining the overall catabolic rate. A 2 to 3-fold difference in digestion rates was observed between the proteins employed as antibody (guinea pig IgG1 or IgG2) and as antigen (DNP-BSA). This finding suggests that the intrinsic susceptibility of ingested proteins to enzymatic hydrolysis may be the prime determinant of digestion rate. As with ingestion, no discrimination was observed in the degradation of complexes of different size or IgG antibody subclass. The observations in this and the preceding study (Eur. J. Immunol. 1980. 10:317) indicate that complex size is important in determining the level of uptake by phagocytes but not subsequent events associated with catabolism.

The binding of soluble immune complexes of guinea pig IgG2 to homologous peritoneal macrophages. Determination of the avidity constants at 4 degrees C.

Soluble immune complexes of guinea pig IgG2 anti-DNP (2,4-dinitrophenyl) antibodies and DNP19BSA (bovine serum albumin) were prepared at a variety of antibody-to-antigen ratios. The mean sizes of the complexes were determined by gel filtration, and equilibrium binding of the complexes to homologous peritoneal macrophages was measured at 4 degrees C. Scatchard plots of the binding data were linear indicating that the complexes were binding to a uniform population of functionally independent membrane receptors. The avidity constants for complex binding to macrophages increases from 15.4 X 10(7) M-1 for complexes containing an average of two antibody molecules to 59.0 X 10(7) M-1 for complexes containing an average of four, compared with an association constant for monomeric IgG2 of 0.21 X 10(7) M-1. Calculation of the molar-free energy changes accompanying monomer and immune complex binding revealed not only that the intrinsic binding activity of mononer was sufficient to account for the complex binding activities by simple cooperation of linked antibodies, but also that the cross-linking of antibodies with antigen imposed a strain on the antibody-receptor interaction, resulting in complex binding energies that were lower than the values expected from a simple cooperative mechanism. Complex inhibition by monomeric IgG2 of unrelated antibody specificity was studied, and it was observed that high concentrations of monomer led to an increase in macrophage discrimination between complexes of different size. The relevance of this finding to the in vivo clearance of circulating complexes is discussed.

Sperm immobilization by sera from unimmunized guinea pigs: requirements for immunoglobulin and complement

The components in serum from non-immunized guinea pigs responsible for the immunological immobilization of human spermatozoa have been elucidated. Heating the serum at 56°C for 30 min, pretreatng the serum with purified cobra venom factor or the inclusion aggregated human IgG in the immobilization test all eliminated its immobilizing capacity, suggesting the involvement of complement in this process. Using purified guinea pigs components, it was established that complement components C1-9 as well as guinea pig IgG were required for immobilization. Rabbit IgG, but not human IgG, could be substituted for guinea pig IgG without affecting the degree of immobilization. Absorption of guinea pig IgG with human spermatoza to its addition to C1-9 and motile spermatoza led to the loss of all immobilizing activity. Thus, it appears that ‘natural’ antibody to spermatoza plus all nine complements are required for sperm immobilization.

Fcγ Receptor of the Murine Macrophage-Like Cell Line P388D1

Abstract Studies of the binding of monomeric murine IgG2a and IgG2b to the macrophage-like cell line P388D1 confirmed the existence of at least two classes of Fc receptors. One receptor binds IgG2a preferentially, whereas the second interacts better with IgG2b. Approximately equal numbers of each class of receptor were present. The receptors showed a marked cross-specificity toward IgG2 subclass proteins but did not recognize murine IgG1. The P388D1 receptors were used to study the phylogenetic emergence, the conservation, and evolution of the molecular structures necessary for the expression of cytophilic activity by IgG. Heterocytophilic activity of plasma, sera, or purified immunoglobulins from modern representatives of the major phylogenetic families was assessed by competitive displacement of specifically bound radiolabeled IgG2a or IgG2b. The P388D1 Fc receptors showed marked species specificity. Fish IgM and non-IgM 7S immunoglobulins from amphibians, reptiles, and avians lacked cytophilic activity toward P388D1; only turtle plasma could displace murine IgG2 proteins from their receptors. In higher vertebrate species, rat, rabbit, and human IgG competed for the two classes of receptor equally well. In contrast, both guinea pig IgG1 and IgG2, and also IgG from bovine, sheep, goat, horse, and opossum lacked the ability to interact with P388D1. Canine IgG could displace murine IgG2a but not IgG2b from their receptors. These results, when compared to the data available on macrophage receptors for homologous and heterologous IgG in most of the above species, indicate that cytophilic structures have diverged in parallel with the cell-surface receptors and that mouse, rabbit, and human macrophage Fc receptors are structurally related.

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.

A study of the synaptogenesis in the cerebellar cortex through chronic treatment and immunocytochemistry of β‐Bungarotoxin

The mechanism of synaptogenesis between dendritic spines of Purkinje cells and parallel fibers in the chicken cerebellum was studied through chronic treatment and immunocytochemistry of beta-Bungarotoxin (beta-BT). Attention was directed to the question of whether the presynaptic specializations (presynaptic vesicular grids composed of presynaptic dense projections and associated vesicles) of the parallel fibers can differentiate in the absence of the Purkinje cells. Normal cerebella from 18-day and 21-day chick embryos, incubated with beta-BT and reacted with HRP-labeled anti beta-BT guinea pig IgG, showed a positive HRP reaction on Purkinje cells but not on external and internal granule cells. Thus, in chicken cerebellum, beta-BT primarily affects Purkinje cells. When beta-BT was applied to chick embryos at 3-day intervals, beginning on the 4th day of incubation, the cerebella were markedly reduced in size and most of the Purkinje cells as well as the nerve fibers in the white matter disappeared between the 18th and the 21st day of incubation. Folia of the 21st day experimental cerebella were irregular in shape and the area of the midsagittal section was one fourth that of the controls. In the 21st day cerebella treated with beta-BT, the majority of Purkinje cells disappear. However, the external granule cells remain intact and showed the usual mitotic activity. The majority of the inner granule cells were normal. Some of the parallel fibers, which display presynaptic vesicular grids, established synaptic contact with stellate cells and the dendritic spines of the few Purkinje cells that survived the treatment. However, vast areas of the molecular layer contained neither dendrites of Purkinje cells, nor parallel fibers displaying presynaptic vesicular grids devoid of their postsynaptic counterpart. In such areas, the molecular layer consisted of parallel fibers of uniform diameter, some of which contained accumulations of vesicles, but displaying no presynaptic dense projections. This suggests that parallel fibers may not be able to completely develop the presynaptic vesicular grids in the absence of their target cells.

Electrokinetic study of the reactions of peritoneal macrophages and eosinophils with IgG immune complexes.

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of guinea pig IgG immune complexes on the electrophoretic mobility distributions of guinea pig resident peritoneal cells. The resident population of cells is composed of macrophages (approximately 75%) and eosinophils (approximately 25%). These cells were separated according to the well-established method of Boyum. Populations of resident macrophages, eosinophils, and the unfractionated samples were incubated with soluble immune complexes, antigen alone, or antibody alone. The mean mobility of the resident macrophages decreased approximately 60% when incubated in the presence of immune complexes, although no effect could be discerned in the presence of antigen or antibody alone. The width of the resulting macrophage mobility distribution was larger than that of the control distributions, with a broad shoulder on the high-mobility side, indicating a heterogeneous response of the macrophages to the immune complexes. Eosinophils react in two distinct fashions. One population of eosinophils is present near the control experiments. The second population reacts in a manner very similar to that of macrophages. This suggests that at least two populations of eosinophils are present in the unstimulated guinea pig peritoneal cavity. Results that are intermediate between these two cases are found when unfractionated samples are studied.

Differentiation autoantigen of testicular cells and spermatozoa in the guinea pig

Guinea pigs, immunized with autologous testes, produce antibody that reacts with surface antigen(s) of epididymal sperm and testicular cells. Both IgG and F(ab') 2 fragments of the antibody molecules bind to testicular cells. Only 50% of the testicular cells had detectable surface antigens and they are identified by rosette formation with Staphylococcus aureus, which bind only to testicular cells and sperm coated with guinea pig IgG. Rosette-positive testicular cells (percentage of cells positive) are: early round spermatids (8%), oval late spermatids (98%), cytoplasmic droplets (93%), and heads (and midpieces) of testicular and epididymal sperm (100%). As determined by a quantitative cellular radioimmunobinding assay, antibody to testicular cells is absorbed by testis or sperm but not by other male or female guinea pig tissues. The antigen, which could be multiple, is therefore sperm and late spermatid specific, and is designated testicular cell-sperm differentiation autoantigen. The surface distribution of this antigen is modulated by antibody to exhibit temperature-dependent translational movement along the plasma membrane.

Comparison of four‐layer radioimmunoassay and electron microscopy for detection of human rotavirus

A four-layer radioimmunoassay (RIA) using polystyrene beads as the solid phase, anti-rota guinea pig IgG as primary antibody, anti-rota rabbit IgG as secondary antibody, and 125I-labelled sheep anti-rabbit immunoglobulin as indicator antibody has been developed for the detection of human rotavirus in stool specimens. A comparison was made of the developed RIA, routine electron microscopy, and research electron microscopy of 147 unconcentrated stool specimens from patients with infantile gastroenteritis. In routine electron microscopy 17 (11.6%) false-positive or false-negative results were obtained when compared with research electron microscopy. Each specimen positive in research electron microscopy was positive in RIA, and six additional RIA positives were found from 58 electron microscopy negative specimens. A confirmatory test was necessary to find out marginally positive but nonspecific reactions in RIA. The developed radioimmunoassay is slightly more sensitive than research electron microscopy of unconcentrated stool specimens and considerably more sensitive and more specific than routine electron microscopy.