Equine IgG Research Articles

Specific binding of bovine, ovine, caprine and equine IgG subclasses to defined types of immunoglobulin receptors in gram-positive cocci

One hundred and twenty bacterial strains were tested for non-immune binding of radiolabelled bovine, ovine, caprine and equine immunoglobulins. Bacteria possessing previously defined IgG receptors interacted in a well defined manner with purified IgG subclass immunoglobulins. Human group C and G streptococci carrying IgG receptors type III were capable of binding all IgG subclasses in the four mammalian species studied. Protein A-containing staphylococci demonstrated a restricted specificity with binding of bovine IgG1, ovine IgG1, caprine IgG1 and IgG2 as well as equine IgG(ab). Group A streptococci which can bind human IgG did not show specific reactivity. A new type of binding unrelated to the regular Fc-mediated binding was observed with equine IgG(T). The differences in specificity for IgG subclasses suggest that structures with binding capacity to streptococcal type III Fc receptors are different from staphylococcal protein A reactive sites. Inhibition experiments performed with purified immunoglobulins showed that individual IgG subclasses differed greatly in their inhibiting capacity reflecting differences in avidity. The high avidity and the broad, unrestricted immunoglobulin G reactivity of streptococcal IgG receptor type III indicate that human group C and G streptococci may provide a valuable tool for solid phase absorption of immunoglobulins from several mammalian species.

Equine IgG and IgG(T) antibodies: Dependence of precipitability on both antigen and antibody structure

Equine IgG(T) antibodies formed insoluble complexes with ovalbumin which has multiple, non-repeating determinants, but formed only soluble complexes with proteins bearing 8. 12 or 30 dinitrophenyl (Dnp) determinants. IgG(T) antibodies to Dnp agglutinated erythrocytes coated with Dnp-protein antigens, but not erythrocytes dinitrophenylated by reacting directly with dinitrofluorobenzene. IgG antibodies agglutinated all erythrocyte preparations tested and precipitated with all antigens except Dnp-proteins with 8 Dnp determinants. The equilibrium constant for the intramolecular reaction of IgG antibody with Dnp 12BSA was estimated to be 10 or less.

Isolation of equine IgG(T) by hydrophobic interaction chromatography

Isolation of equine IgG(T) by hydrophobic interaction chromatography

Isolation of equine IgG(T) by hydrophobic interaction chromatography

Hydrophobic interaction chromatography with phenyl-Sepharose CL-4B was used to isolate IgG(T) from the serum of non-hyperimmunized horses. IgG(T) was bound to phenyl-Sepharose CL-4B in the presence of 1 M ammonium sulfate and eluted in 0.6 M ammonium sulfate. In contrast to human IgA, the IgA of various anima species including the horse, is eluted only with ammonium sulfate concentrations below 0.6 M, allowing the purification of IgG(T) without contamination by IgA.

Thermodynamics of antibody-antigen reactions. 2. The binding of bivalent synthetic random coil antigens to antibodies having different antigen precipitating properties

The objects of this study were the equine IgG and IgG(T) classes of antibodies with immunologic specificity for the dinitrophenyl group and bivalent antigens consisting of linear poly(ethylene glycol) polymers which terminated at both ends in dinitrophenyl groups. Complex formation between antibodies of both classes and one of several sharp fractions of antigen having number average molecular weights in the range 25 000 to 75 000 were studied by measuring the light scattered from solutions containing equimolar amounts (approximately 5 x 10(-6) mol/L) of one of the antibodies and one size fraction of antigen, and variable amounts of monovalent hapten. The data were analyzed in the context of a model that accounted for the formation of linear and cyclic complexes of all extents of aggregation. Two parameters in addition to the intrinsic antibody-dinitrophenyl group association constant were found to be necessary in the assumed equilibrium model to account for the behavior of the system. One of these accounted for the looses in configuration entropy that resulted when a random-coil polymer became bound at one end to a space-occupying antibody. The other was a ring closure factor for the formation of cyclic complexes. Ring closure factors for the formation of larger cyclic complexes (present in only small amounts under the conditions studied) were related to the ring closure factor for the formation of the smallest, which was found to increase as antigen size decreased, and for each antigen size to be consistently higher for IgG(T) antibody than for IgG antibody. Comparison of the theoretically estimated values of the two parameters within their measured values indicated that the average conformation of IgG antibodies in solution is open ("T" shaped) but the average inter-Fab are angle in IgG(T) antibodies is approximately 60 degrees or less.

Thermodynamics of antibody-antigen reactions. 1. The binding of simple haptens to two classes of antibodies fractionated according to affinity.

The thermodynamic parameters which characterize the binding of dinitrophenylglycine and dinitrophenylmethoxypoly(ethylene glycol) to selected affinity classes of equine IgG and IgG(T) antibodies were determined by fluorescence quenching and flow calorimetry. The binding enthalpies and entropies were in all cases large and negative, falling in the ranges -14 to -17 kcal/mol and -18 to -25 eu, respectively. The differences in the enthalpies and entropies of binding for different affinity classes and for different haptens are discussed with reference to differences in the structures of the haptens studied and as indications of differences in binding site structure. In addition, the apparent existence of fluorescent side chains which can transfer energy to either hapten binding site in IgG(T) antibodies but not in IgG antibodies is interpreted as indicative of a smaller average interbinding site distance in IgG(T) than in IgG antibodies.

Identification of Haemoglobin S in red cells and normoblasts, using fluorescent anti-Hb S antibodies.

Anti-haemoglobin S antibodies were raised in horses and purified by affinity chromatography. These antibodies recognize beta6 valine, while they fail to bind to haemoglobins with a glutamy1 (Hb A) or a lysy1 (Hb C) residue in this position. The purified anti-Hb S antibodies were composed of equine IgG(a, b) and IgG(T) subclasses and failed to cause precipitation with Hb S, evidently because of the bivalencey of both antibodies and antigen. Identification of Hb S in single erythrocytes was achieved by reacting the fluorescein isothiocyanate-conjungated anti-Hb S antibodies with the haemoglobin antigen in fixed blood smears. All the red cells from persons with sickle cell trait and sickle cell anaemia are labelled with the anti-Hb S-FITC, while red cells from persons with normal haemoglobin or other mutant haemoglobin genotypes are not. Erythrocytes from newborns with sickle cell trait bind the anti-Hb S antibody, although the intensity of labelling varies from cell to cell. This method of cellular identification of Hb S preserves the morphological characteristics of the reactive cells and permits observations of the pattern of haemoglobin synthesis among cells of various degrees of maturity. Fluorescent identification of Hb S in fixed blood smears may be applicable to prenatal diagnosis of haemglobinopathies.

Mixed IgG-IgA cryoglobulinemia in human serum sickness. Evidence for Equine IgG in the cryoprecipitate.

Serum sickness followed the administration of anti-lymphocyte globulin to a patient with multiple sclerosis. In addition to other characteristic features of this syndrome, there was hypocomplementemia and transient renal dysfunction similar to that observed in the 'one-shot' experimental model of serum sickness. Cryoglobulinemia was transiently demonstrable at the height of the inflammatory response. Analysis of the purified cryoprecipitate revealed the presence of human IgG and IgA, and, in addition, equine IgG. This demonstration of a well-defined exagenous antigen in the cryoprecipitate provides further support for the immune complex nature of mixed cryoglobulins.

The Metabolism of lgG(T) in the Newborn Foal

Summary The distribution and metabolism of IgG, the predominant immunoglobulin in equine colostrum, was studied in 7 newborn foals, using 125I-labelled IgG. An intravascular/extravascular ratio of 1/1·0 was found. Equilibration was apparently complete by 51 h. The apparent plasma half-life of the labelled immunoglobulin was 23 days. More precise quantitation of the absorption of equine IgG from the colostrum is now possible.

The Metabolism of IgG in the Newborn Foal

Summary The distribution and metabolism of IgG, the predominant immunoglobulin in equine colostrum, was studied in 7 newborn foals, using 125I-labelled IgG. An intravascular/extravascular ratio of 1/1·0 was found. Equilibration was apparently complete by 51 h. The apparent plasma half-life of the labelled immunoglobulin was 23 days. More precise quantitation of the absorption of equine IgG from the colostrum is now possible.

Identification and quantitation of equine serum and secretory immunoglobulin A.

Immunoglobulin A (IgA) was demonstrated in equine serum and secretions. This immunoglobulin had a molecular weight extending from 150,000 to 700,000 and reacted with specific antihuman alpha-chain antiserum. Antigenic determinants specific for secretory IgA were demonstrated and found to be absent on serum IgA. Antigen binding activity was detected in IgA from tears. Purified IgA was antigenically distinct from equine IgG, IgM, IgG(T), and aggregating immunoglobulin. Quantitative studies demonstrated that IgA was the predominant immunoglobulin in tears and milk but not in colostrum. The electrophoretic mobility, size, presence of secretory component, and reaction with specific antihuman alpha-chain antiserum demonstrates that this immunoglobulin is the equine homologue of human IgA.

Studies on equine immunologlobulins—III Antigenic bacterial interrelationships among horse and dog γG globulins

1. 1. Antisera prepared against equine IgG globulin, its Fab, Fc fragments and L chains were employed in immunodiffusion analysis to examine their cross reactivity with canine IgG a,b globulins and a canine IgG d paraprotein. 2. 2. Interspecies antigenic similarities were found and cross reactive groupings assigned to various parts of the canine γG-globulin molecule. 3. 3. Immunodiffusion and cross absorption studies with anti-equine IgG, showed canine IgG a,b and IgG d to share some antigenic determinants in common with equine IgG and IgB absent from equine IgG(T).

Carboxy-Terminal Amino Acid Sequences of Canine Immunoglobulin G Subclasses

Abstract The carboxy-terminal octadecapeptides from canine heavy chains of 1) IgGa, b (from normal serum) and 2) IgGd (from a paraprotein) were isolated. The amino acid sequences of both octadecapeptides were determined. Both peptides are clearly homologous to the same heavy chain region of IgG's from other mammals. Previously it has been shown that canine IgGd and equine IgG (T) share certain biologic properties; however, comparison of the sequences of their C-terminal octadecapeptides reveals no uniquely shared residues which would support a close phylogenetic relationship.

Metabolism of Immunoglobulin-G in the Horse

The metabolism of immunoglobulin classes has been closely examined in several animal species. Although the horse has received much attention in experimental and applied immunology there seems to be little information available on immunoglobulin kinetics in this species. The present report describes the metabolism of equine IgG in 4 healthy, normoimmunoglobulinaemic horses, in 1 horse with hyperimmunoglobulinaemia and in 1 horse with relatively low immunoglobulin levels.

Preventing Antibody Formation to Anti-Lymphocytic Globulin in Man

Abstract One or more doses of equine IgG either as anti-lymphocytic globulin (ALG) or deaggregated normal equine IgG (DEGG) were given to 73 individuals including 43 potential or actual allograft recipients, 10 patients with leukemia and 20 normal volunteers in order to define conditions which would prevent formation of detectable antibodies to horse IgG in patients being treated with ALG. Formation of antibody was measured by studying the plasma clearance of radioactively-labeled horse IgG and by an immunoadsorbent assay. DEGG and ALG were equally immunogenic when either was given alone, causing antibody formation to horse IgG in 20 of the 27 patients (74%) treated with one or the other agent. Moreover, treatment of patients with ALG given along with maximum tolerated doses of azathioprine and methyl prednisolone did not reduce the incidence of antibody formation to horse IgG. In contrast, 8 of 27 patients (30%) who were given chemical immunosuppressive treatment for at least 2 days followed by 2 to 18 daily intravenous injections containing 50 µg/Kg of DEGG developed detectable antibodies to horse IgG when subsequently treated with ALG. Of these 8, only 1 developed rapid immune elimination of labeled horse IgG. These results suggest that it is now possible to evaluate the toxic effects and therapeutic efficacy of ALG in man free of those manifestations which might otherwise be ascribed to allergic reactions.

An Examination of the Cross Reactivity of Human and Equine γG Globulins and Their Fragments by Immunodiffusion

Summary Rabbit antisera prepared against equine γG globulin, Fab, Fc, H and L chains were employed in immunodiffusion analysis to examine their cross reactivity with antigens related to various portions of the human IgG molecule. Antigenic similarities between the corresponding H and L polypeptide chains of both species were found and cross reactive groupings assigned to locations within chains. A collection of 23 Bence Jones proteins examined for cross reactivity with several rabbit anti-equine Fab and L chain reagents showed cross reactivity of human L chains to be associated principally with λ type proteins. Immunodiffusion analysis of reciprocal cross reactions involving the ability of several rabbit anti-human IgG reagents to precipitate with antigens related to various portions of the equine IgG molecule showed cross reactivity to be associated with determinants on equine Fc and F′ fragments. Interspecies antigenic similarities between equine L chains and type λ human L chains was also demonstrated by the cross reaction shown by an anti-type λ Bence Jones protein reagent with equine L chains.