Porcine IgG Research Articles

Application of radioimmunological methods for checking the quality of class-specific antibodies against bovine and porcine immunoglobulins

Class-specific antibodies against bovine IgG1, IgG2, IgM and IgA and porcine IgG, IgM and IgA immunoglobulins were prepared. Their class specificity was assessed by two radioimmunological methods, namely, radioimmunoelectrophoresis and double antibody sandwich radioimmunoassay. The methods are highly specific and sensitive and do not require the use of purified immunoglobulins, but can be performed with normal serum or colostrum. It was confirmed that antibodies found satisfactory in these tests were suitable for a wide range of use including radioimmunoassay and enzyme linked immunosorbent assay.

Immunohistochemical studies of the local immune system in the reproductive tract of the sow

Tissues from the reproductive tract of sexually mature female pigs were examined histologically using antisera to porcine IgG, IgA and IgM coupled to horseradish peroxidase. Plasma cells synthesising all three immunoglobulins were found in all regions of the tract, with IgA-producing cells as the predominant type. Larger numbers of plasma cells were found in the lower region of the tract and the number of plasma cells was raised during oestrous. Intercellular staining of immunoglobulins was also observed and was also under hormonal influence. The results indicate the presence of a local mucosal immune system in the female reproductive tract of the pig.

Proteolytic activity as a regulator of the transmission of orally fed proteins from the gut to the blood serum in the suckling rat.

14-day-old rats were orally fed with porcine colostrum or serum having a low or high activity of protease inhibitors (i.e., sow colostrum trypsin-chymotrypsin inhibitor or soybean trypsin inhibitor). The uptake of undigested porcine albumin and IgG to the blood serum of these rats was studied 4 h after feeding. The effect of the exclusion of proteases to the intestinal lumen by means of pancreatic duct ligation prior to feeding was also studied. The results from these feeding experiments in the presence of protease inhibitors or in the absence of pancreatic proteases agreed well. It was concluded that for the sucking rat the intraluminal proteolytic activity in the gastrointestinal tract is a regulator for nonselective protein uptake, as represented by albumin, while the selective absorption of IgG was unaffected.

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.

Measurement of staphylococcal protein A and detection of protein A-carrying staphylococcus strains by a competitive ELISA method

A competitive ELISA for the estimation of staphylococcal protein A is described. Tetanus toxoid is insolubilized on polystyrene and incubated with human antitoxin, which renders the Fc-piece of this antibody freely accessible to protein A. The binding of the latter is demonstrated by its competition with protein A-phosphatase conjugate. The method has been shown to be sensitive and reproducible. It has been used for the detection of protein A in culture supernatants and on living pathogenic staphylococci. The test could therefore be of diagnostic value. Protein A is also present in extracts of food contaminated with enterotoxic staphylococci. It can be eliminated by a simple absorption with insoluble porcine IgG.

The interactions of porcine and ovine, serum and colostral immunoglobulins with staphylococcal Protein A.

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produce by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG1, IgG2, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG1 from most ewes possessed a low affinity for Protein A whereas ovine IgG2 generally possessed a high affinity; 100% of the IgG2 in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.

The placental Fcgamma receptors studied using immune complexes of peroxidase.

Immune complexes of horseradish peroxidase (HRP) and rabbit IgG antibodies to HRP were used to study the Fcgamma receptors in normal human placenta. Cryostat sections of placental tissue were incubated with the complexes, and the peroxidase activity was revealed histochemically. The bound complexes were localized to the apical surface of the trophoblasts and endothelial cells of the fetal stem vessels. Binding also occurred within the wall of some fetal vessels, to stromal cells and occasionally to areas corresponding to the trophoblastic membrane. The strongest binding was obtained with immune complexes prepared at slight antigen excess. Eight- to sixteen-fold increased concentration of human and rabbit IgG was needed to block the binding of immune complexes. Bovine and porcine IgG did not block the binding. Treatment of tissue sections with neuraminidase enhanced the binding activity of the receptors. The technique is very convenient for studies of Fcgamma receptors in tissue. However, unfixed frozen placental tissue was not suitable for ultrastructural studies.

Interactions of porcine IgG and porcine lymphocytes with protein-A Sepharose

Interaction of Protein-A Sepharose with porcine IgG showed that the binding capacity for porcine IgG was slightly lower than for human IgG. A small proportion of antibody activity of porcine serum was not reactive with Protein-A Sepharose. The interaction occurred with Fc fragments obtained either by papaĭn or by trypsin splitting of IgG but not with Fc' fragments. However, Fab fragments obtained after papaĭn or trypsin hydrolysis as well as Fab' 2 and Fab' fragments obtained by pepsin cleavage were partly retained onto the Protein-A Sepharose column. No antigenic differences were observed between retained and non-retained Fab, Fab' and Fab' 2 fragments with specific anti-porcine light chain antiserum. Thus, the interaction between Protein-A from S. aureus and porcine IgG is mediated not only through the Fc fragment, but also partly through the Fab part of the molecule. The resolubilized heavy chains did not bind to Protein-A Sepharose, suggesting that structural requirements are necessary for recognition. A subpopulation of porcine lymphocytes could be adsorbed onto Protein-A Sepharose beads, a binding specifically inhibited in the presence of porcine IgG.

Identification and characterization of three immunoglobulin classes in the Northern Fur Seal Callorhinus ursinus

Three immunoglobulin classes were isolated and identified from serum pools and throat mucus of the Northern Fur Seal, Callorhinus ursinus. These classes were shown to cross-react with antigenic determinants of human IgM and IgA and porcine IgG. Data obtained from gel filtration, ultracentrifugation, and reduction experiments indicate that these immunoglobulin classes have physiochemical properties similar to those in terrestrial mammals. Fractionation of serum on DEAE anion exchangers demonstrated two distinct peaks of IgG, the first of which (designated IgG-fp) possessed an antigenic determinant not found on the second peak (IgG-sp). On this basis, we conclude that the two peaks represent subclasses of IgG.

Endocytosis and immunoglobulin transport across the small intestine of the new-born pig.

1. Measurements of porcine and human IgG transport across the small intestine of the new-born pig have been made in vivo and related to the amount of endocytosis taking place.2. The amount of immunoglobulins transported, following intraduodenal administration to conscious animals, is directly related to the degree of endocytosis which these proteins produce. Administration of protein in amounts sufficient to cause a maximal endocytotic response causes saturation of the protein transporting process.3. Absorption of small amounts of human IgG from the small intestine can be accelerated by the addition of sow colostrum or porcine IgG in quantities sufficient to stimulate endocytosis. These effects disappear when the amount of human IgG administered is itself sufficient to fully stimulate endocytosis.4. Preferential transport of porcine over human IgG can be demonstrated when both are given as a single solution to individual pigs. The degree of preference is, however, small in relation to the total amount of immunoglobulin transported.5. The initial formation of endocytotic vacuoles in the pig intestine seems, unlike the situation in rats and mice, to provide a major route for the trans-cellular movement of macromolecules including immunoglobulins. Present evidence suggests that, though sow colostrum can on occasion stimulate such transport, it does so merely by acting as a protein-containing solution to increase the amount of endocytosis taking place.

Biosynthesis of the carbohydrate units of immunoglobulins. 1. Purification and properties of galactosyltransferases from swine mesentary lymph nodes.

Galactosyltransferase (UDPgalactose:glycoprotein galactosyltransferase EC 2.4.1.22) was isolated from swine mesentary lymhromatography on Sepharose 4B colums containing covalently bound p-aminophenyl-beta-D-N-acetylglucosamine. The homogenous enzyme showed a single band on disc gel electrophoresis and had a specific activity of 35 nmol min-1 (mg of protein)-1 at 37 degrees C. A molecular weight of 57 000 was obtained by exclusion chromatography, sucrose density centrifugation, and sodium dodecyl sulfate-gel electrophoresis. The same molecular weight was obtained after reduction and alkylation which indicates that the enzyme is composed of only a single polypeptide chain. The enzyme catalyzed the formation of beta1 leads to 4 bonds between galactose and free terminal N-acetylglucosaminyl residues of soluble preparations of porcine IgG immunoglobulin heavy chain, fetuin, ovalbumin, and ovomucoid. An endogenous glycoprotein, present in particulate subcellular preparations, was also a very good substrate for the enzyme, and it was identified as incomplete IgG immunoglobulin heavy chain. The Km of the purified enzyme was 2.9 x 10(-5) M for fetuin, 5.4 x 10(-5) M for ovalbumin, 2.0 x 10(-5) M for IgG immlnoglobulin heavy chain, and 2.2 x 10(-5) M for UDP-galactose. About 20% of the total galactosyltransferase activity in lymph node homogenates was present in the cytosol fraction, and 80% was in the microsomal and Golgi fractions. The kinetic properties of the bound and soluble galactosyltransferases were similar,and both required Mn2+ for maximal activity. However, the bound enzyme required the addition of detergent, lysolecithin, GDP-mannose, and UDP-N-acetylglucosamine for maximum activity. These compounds did not influence the activity of the soluble transferase. The membrane preparations catalyzed the transfer of galactose from UDP-galactose and N-acetylglycosamine from UDP-N-acetylglucosamine to incomplete oligosaccharide chains of endogenous IgG immunoglobulin bound to these particles. The labeled products of these reactions were isolated, and the structures of their oligosaccharide chains were determined and compared with those isolated from the heavy chain of porcine IgG immunoglobulin. The glycopeptide prepared from the endogenous acceptor and the major glycopeptide prepared by proteolytic digestion of the heavy chain of porcine IgG immunoglobulin has identical structures. The following structure for the carbohydrate chains of porcine IgG immunoglobulin was determined by sequential enzymatic hydrolysis and methylation studies.

Heat sensitivity of porcine IgG

The sensitivity to heat of porcine IgG was studied. The serum from immunized pigs was heated at 56°C for 30 min as for decomplementation. The elution pattern of the serum proteins on an agarose gel column showed a dramatic change with the appearance of a large peak of the gel-excluded material. This peak contained mainly IgG molecules which still remained its antibody activity. This fact points to misinterpretations which can easily occur in 7S and 19S antibody recognition during the porcine immune response. Correlation is suggested of this property with the large number of interheavy chain disulfide bridges present in porcine IgG.

Different types of precipitating antibodies in early and late porcine anti-dinitrophenyl sera

The anti-dinitrophenyl antibodies produced in pigs at early and late phases of immune response show striking differences when interacting with multivalent antigens. While the early antibody is capable of precipitating hapten-protein conjugates in a very broad range of antigen concentrations, the late antibody precipitates only in a narrow zone of antibody excess. The precipitability of the late antibody is more sensitive to antiserum dilution and to heat treatment than the precipitability of the early antibody. Both antibodies have a sedimentation coefficient 6·7 S and behave as antigenically identical in immunodiffusion. An evidence of certain structural differences between the early and late antibodies is obtained from the patterns of radioactively alkylated peptides containing inter-chain half-cystine residues of the heavy chains. The contrasting character of antigen precipitation can hardly be explained assuming that an increased association constant is the only difference between early and late antibodies. A possibility is suggested that the early and late antibodies represent closely related subclasses of porcine IgG differing by the shape and/or flexibility of the molecule.

Subclasses of porcine immunoglobulins G. Production of subclass specific antisera with S-sulfonated gamma chains.

AbstractUsing antisera made against porcine S‐sulfonated heavy chains two subclasses of porcine IgG could be distinguished on the basis of antigenic determinants located on the Fc fragment. The antisera prepared against the porcine gamma chains reacted with other mammalian IgG proteins, but not with avian, amphibian or fish immunoglobulins. Antigenic determinants responsible for cross‐reactivity are further localized on the Fc′ fragment of the porcine IgG molecule. Comparable antisera, specific for porcine light chains, did not cross‐react with IgG from any of the other species tested. Porcine IgG and F(ab′)2 fragment reacted with both the anti‐heavy and anti‐light chain sera, while the Fab fragment reacted only with the anti‐light chain sera. Important antigenic determinants are thus located on the light chains and the Fc portion of the heavy chain of the porcine IgG molecule.

Comparison of Various Avian and Mammalian IgG Immunoglobulins for Salt-Induced Aggregation

Abstract Salt-induced aggregation of chicken, pheasant and quail IgG has been demonstrated by ultracentrifugation and gel filtration. Avian IgG immunoglobulins had sedimentation coefficients of 11–14S in 1.5 M NaCl (high salt), and 7S in 0.15 M NaCl. The aggregation of chicken IgG was reversible. Rabbit, porcine, bovine, sheep and human IgG immunoglobulins did not form aggregates in high salt. The Fc fragments of chicken and pheasant IgG were precipitated by high salt, whereas the Fab fragments were not aggregated by high salt. The salt-induced aggregation of chicken IgG antibodies was suggested as an explanation for the enhanced precipitation of chicken IgG antibodies by antigen in high salt.