Binding Characteristics Of Antibodies Research Articles

Diffuse Cutaneous Leishmaniasis Acquired in Peru

A case of diffuse cutaneous leishmaniasis (DCL) acquired in Peru is described. The causative agent was Leishmania mexicana amazonensis as determined by isoenzyme analysis and species-specific monoclonal antibody binding characteristics. Histological examination of biopsy material showed a large number of intracellular and extracellular amastigotes and few lymphocytes. Treatment with meglumine antimoniate (Glucantime) administered iv at a dosage of 20 mg antimony/kg body weight/day for 60 days resulted in visible improvement of the lesions, but not in clinical or parasitological cure.

The peritoneal Ly‐1 (CD5) B cell repertoire is unique among murine B cell repertoires

Ly-1 (CD5) B cells and conventional B cells represent two distinct lineages of murine B cells which are distinguishable by expression of surface molecules, organ location, ontogeny and development and antibody production in vivo. In order to assess whether the different developmental pathways of Ly-1 B cells and conventional B cells result in different antibody repertoires, we have used limiting dilution analyses to determine frequencies of B cells making antibodies capable of binding to a range of antigens including haptens, proteins, bacterial polysaccharides and bromelain-treated mouse red blood cells. Starting populations of B cells were purified from spleen, peritoneum and bone marrow of adult BALB/c mice or from spleens of newborn mice by use of the fluorescence-activated cell sorter. The peritoneal Ly-1 B cell repertoire was found to be different from that of conventional B cells, with between 5- and 100-fold higher frequencies of clones producing IgM antibodies capable of binding to the antigens tested. However, when tested, the majority of Ly-1 B cell anti-haptenic antibodies did not show the high affinity binding or fine specificity characteristics of specific antibodies elicited in immune responses in vivo. The high frequencies of antigen-reactive antibodies within the Ly-1 B repertoire are most likely explained by the presence of clones secreting low-affinity or multireactive antibodies. The Ly-1 B cell repertoire is not mirrored in repertoires from either newborn B cells or virgin B cells in adult bone marrow. Therefore, either Ly-1 B cells develop from distinct precursors with intrinsically different mechanisms of V gene usage and recombination, or newly formed Ly-1 B are heavily selected on specificity for entry into this peritoneal lineage. If the second alternative is true, bacterial antigens in the gut are not required for selection of this unique repertoire, as Ly-1 B cells in germ-free mice also show the multireactive repertoire characteristic of this B cell lineage in normal mice.

Adsorption equilibrium in immunoaffnity chromatography with antibodies to synthetic peptides

The effects of charged residues in peptide antigens on the binding characteristics of polyclonal antipeptide antibodies were studied using immunoadsorbents prepared by coupling the antibodies to CNBr-activated Sepharose 4B. Among the antipeptide antibodies, an antibody to the peptide without charged residues showed the most stable interaction with the peptide to the changes in pH. Conversely, the binding affinity of antibodies to the pep-tides with histidine residues having a unique pKa value of 6.0 decreased steeply with pH at around 6.0. The binding affinity of an antibody to the peptide with many charged residues decreased steeply with an increase in the ionic strength (adjusted by NaCl). Since circular dichroism (CD) spectrum measurements indicate that these peptides show disordered structures in the pH range of adsorption measurement, the dependence of peptide-antibody interaction on environmental conditions is attributed to the characteristics of side chains of the peptides. These results indicate that the dependence of the binding affinity of antipeptide antibodies on pH and the ionic strength is dominantly affected by the number and the pKa values of charged residues in the peptides.

Method validation in the bioanalytical laboratory

Bioanalytical methods, based on a variety of physico-chemical and biological techniques such as chromatography, immunoassay and mass spectrometry, must be validated prior to and during use to engender confidence in the results generated. The fundamental criteria for assessing the reliability and overall performance of a bioanalytical method are: the evaluation of drug and analyte stability, selectivity, limits of quantification and detection, accuracy, precision, linearity and recovery. The extent to which a method is validated is dependent on its prospective use, the number of samples to be assayed and the use to which the data are put. Specific analytical techniques may require additional validation such as antibody-binding characteristics, peak purity determination, evaluation of matrix effects or structural confirmation of the analyte. Ideally each assay should be cross-validated with a method utilizing a highly specific detector such as a mass spectrometer. Once in use, the performance of the method should be monitored using quality control standards. If a method is set up in another laboratory, the performance of that assay should be monitored with quality control standards sent from the originating laboratory.

Importance of pH in defining the specificity and clinical utility of monoclonal antibodies — an evaluation of two anti-CA 19-9 monoclonal antibodies

The effect of pH on the binding characteristics of monoclonal antibodies against tumor associated antigens has been studied, using anti-CA 19-9 monoclonal antibodies as an example. The 1116-NS-19-9 monoclonal antibody is used in several commercially available two-step sandwich IRMAs for the measurement of the CA 19-9 antigen in cancer patient sera (Centocor CA 19-9 RIA, Malvern, PA, U.S.A., or similar kits by Centocor licensees). The B25.10 monoclonal antibody is in use in the Truquant ® GI ™ RIA (Biomira Inc., Edmonton, Alberta, Canada), a competitive inhibition assay measuring the CA 19-9 antigen. We determined the K a value of the B25.10 and the 1116-NS-19-9 antibodies at pH 4.5 and 7.3 on a solid phase coated with CA 19-9 bearing mucin. The 1116-NS-19-9 antibody had a K a value of 4.1 × 10 8 at pH 4.5 and approximately 0.3 × 10 8 at pH 7.3. The K a value of B25.10 was in the range of 0.7 – 4.1 × 10 9 at either pH, but some-what lower at pH 4.5. At pH 4.5 (the pH optimum for binding of 1116-NS-19-9 to the CA 19-9 antigen [Del Villano, B.C., 1984]) the binding of 125I-labeled B25.10 to the solid phase was comparably inhibited by 1116-NS-19-9 and B25.10. However, at neutral pH (pH 7.2) the apparent affinity of the 1116-NS-19-9 antibody for the CA 19-9 antigen is significantly diminished [Del Villano, B.C., 1984] while that of B25.10 is slightly higher. Consequently, the ability of the 1116-NS-19-9 to inhibit the binding of the B25.10 antibody to the CA 19-9 solid phase is greatly reduced in the physiological pH range. The consequences of these findings for in vivo applications are discussed.

Changes to desmosomal antigens and lectin-binding sites during differentiation in normal human epidermis: a quantitative ultrastructural study.

During epidermal differentiation, desmosomes undergo a series of changes in their abundance, structure and properties, which has previously been defined by conventional electron microscopy and the use of antibodies to desmosomal proteins at the light-microscope level. Such changes in a major adhesive organelle would be expected to have a significant role in the maintenance of epidermal organization, and therefore require more detailed characterization. In the present study, modifications to certain desmosomal components in normal human epidermis have been located and quantified by immunogold electron microscopy. Antibodies to desmosomal protein dp3 and glycoprotein dg1 were used to label the cytoplasmic regions of the junctions and lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) to probe the extracellular glycosylated material. Binding was measured at histologically defined levels and expressed as gold particles per microns of desmosome length (linear particle density: LPD). In addition, desmosome frequency, expressed as the percentage of the cell membrane length occupied by desmosomes, was measured. Highly significant changes in desmosome frequency, diameter and LPD were observed between epidermal strata and, in basal and upper horny cells, between different regions of the same cell surface. These parameters rose to a maximum in the spinous or granular layers: their subsequent decrease continued without interruption across the interface between the living and terminally differentiated horny layers. Remaining reactivity with antibodies, but not lectins, was almost completely abolished immediately before the final disintegration of the desmosome structure in the lower horny layer. In contrast, numerous large, highly immunoreactive desmosomes were retained up to the outer surface in the grossly thickened horny layer found in callus. Though the overall pattern of a rise followed by a fall was similar for all parameters measured, differences were observed between probes. Thus, the extent of the rise in available antigen between the lateral and apical surfaces of the basal cell was greater for dg1 than for dp3; the subsequent decrease in dp3 antigens in upper epidermal layers was more rapid than that for dg1, and changes to both antigens preceded those to lectin-binding sites. These results show that differences in desmosome frequency and in the size and antibody-binding characteristics of individual junctions underlie the heterogeneous distribution of desmosomal components within epidermis that is found by light-microscope immunocytochemistry. They further suggest that the disintegration of desmosomes within normal horny layer, which is an essential preliminary to desquamation, is the culmination of a sequence of events that begins in the upper living tissue and initially involves cytoplasmic components.(ABSTRACT TRUNCATED AT 400 WORDS)

Differences between ocular and generalized myasthenia gravis: binding characteristics of anti-acetylcholine receptor antibody against bovine muscles

We studied the binding characteristics of anti-acetylcholine receptor antibody (AChR Ab) in sera of nine patients with myasthenia gravis (MG) using affinity-purified extraocular muscles (EOM) and foot muscles (FM) of bovine species. The titer of AChR Ab measured with EOM was the same as that measured with FM for both ocular and generalized MG. In patients with ocular MG, the affinity of AChR Ab, determined by Scatchard analysis, was higher for FM than for EOM ( P < 0.05), whereas it was the same for EOM and FM in those with generalized MG. On the other hand, the affinity of AChR Ab for FM was lower in generalized MG than in ocular MG ( P < 0.05), but that for EOM did not differ between the two types of MG. The affinity of AChR Ab did not change after passing the patients' sera through an EOM-affinity column. The EOM-affinity column treatment of the sera decreased the AChR Ab titer measured with EOM in all patients, but the AChR Ab titer measured with FM was decreased in only some of the patients. These data indicate that there are polyclonal or heterogenous AChR Abs in the sera of MG patients, which does not help to explain the clinical difference between ocular and generalized MG.

Specific binding characteristics of high affinity monoclonal antidigitoxin antibodies.

The specificity of various monoclonal antidigitoxin antibodies was characterized using 6 cardiac glycoside analogs. Spleen cells from BALB/c mice, immunized with BSA- or KLH-digitoxin conjugates, were fused with NS1 myeloma cells, and antibody-producing hybrids were identified by radioimmunoassay. Twenty-one monoclonal antidigitoxin-specific antibodies were obtained, 10 of which were cloned and characterized for affinity and specificity. All the antibodies had a high affinity constant, ranging from 8.10(8) to 2.5.10(10) 1/M. On the basis of their binding specificities, the antibodies could be classified into 3 groups: the first contained 7 antibodies exhibiting high cross reactivity (42-100%) with digitoxigenin, whereas the second and third groups did not recognize this analog (cross-reactivity of 1%). In the former group, the absence of the sugar moiety only slightly affected the binding reaction, although for the two other groups, this structure did appear to be involved in antibody recognition. Changes in the functional groups of the hapten molecule led to considerable changes in the antibody-antigen reaction. For all the antibodies except one, saturation of the lactone ring considerably affected binding. These results demonstrated that monoclonal antibodies of different specificities with respect to both the steroid backbone and the sugar moiety of digitoxin can be induced using a digitoxin-protein conjugate.

Isozymes of dog heart Na+-K+-ATPase are immunologically similar to isozymes in brain.

The sodium pump, Na+-K+-ATPase, possesses two populations of cardiac glycoside-binding sites in cardiac tissue. This has been observed in other tissues such as brain where the two sites have been assigned to isozymes of Na+-K+-ATPase termed alpha and alpha +. In a previous study [Am. J. Physiol. 248 (Cell Physiol. 17): C247-C251, 1985], we were unable to demonstrate the presence of an alpha +-form in guinea pig heart sarcolemmal membranes using antibody probes. In the present study, using similar methodology, we show that dog, but not rat or guinea pig, sarcolemmal membranes contain two immunologically distinct alpha-subunits. The antibody-binding characteristics of dog heart alpha and alpha + are similar to the forms found in brain. The relative abundance of the two isozymes, estimated by labeling the sarcolemmal membranes with fluorescein 5'-isothiocyanate, was about equal. We conclude that the two populations of ouabain-binding sites in dog heart may result, at least in part, from the presence of the two isozymes of Na+-K+-ATPase in cardiac tissue of this species.

Improvement in the antibody binding characteristics of microtitre wells by pretreatment with anti-IgG Fc immunoglobulin

A preliminary coating of the surface of polystyrene microtitre wells with anti-IgG Fc immunoglobulin improves the binsing capacity of the wells for antibody and leads to a marked improvement in the reproducibility of immunoassays carried out in the wells. In an enhanced chemiluminescent enzyme immunoassay for oestradiol the binding capacity for oestradiol of wells precoated with anti-IgG Fc immunoglobulin and then coated with anti-oestradiol immunoglobulin was more than twice that of wells coated directly with anti-oestradiol immunoglobulin. Intra-assay precision in the precoated wells ranged from 1.3 to 4.0% compared to 2.9 to 6.7% (0–3200 fmol oestradiol) in wells which had not been precoated.

The IgG subclass distribution of thyroid autoantibodies

There have been several conflicting reports concerning the subclass distribution of thyroid autoantibodies. We have therefore reinvestigated this with both a radioimmunoassay and an ELISA based on the use of a variety of monoclonal antibodies. Our results demonstrate the presence of subclasses IgG 1, IgG 2, IgG 3 and IgG 4 in both thyroglobulin and microsomal autoantibodies from patients with either Graves' disease or Hashimoto's thyroiditis. However, in many patients there is over-representation of the IgG 4 subclass. We also found marked differences in the binding characteristics of monoclonal antibodies directed against the same subclasses, underlining the need for appropriate selection of such monoclonal reagents in any assay.

Evidence that the reduced surface antigenicity of developing Schistosoma mansoni schistosomula is due to antigen shedding rather than host molecule acquisition

Antibody and lectin binding characteristics of Schistosoma mansoni schistosomula maturing in vivo and in vitro were quantitatively assessed and compared in order to investigate the basis of the reduced surface antigenicity of host derived larval schistosomes. Quantitative indirect immunofluorescence assays showed that schistosomula recovered from mice at 24 h and 5-10 days post infection bound low or insignificant amounts of a variety of anti-schistosome antibodies including those from chronically infected and radiation attenuated cercariae-vaccinated mice, a vaccinated rabbit and rabbits hyper-immunized with non-living larval and adult schistosome antigen preparations. In contrast, parasites maturing in vitro continued to bind highly significant levels of each of these antibody preparations until at least 10 days post transformation. To investigate the basis of the decreased surface antigenicity of parasites maturing in vivo, 6-day-cultured parasites were injected intravenously into mice and recovered from the lungs at various times thereafter and examined for their ability to bind both anti-parasite and anti-host antibodies. After 30 min in vivo, cultured schistosomula exhibited a significantly decreased capacity to bind anti-parasite antibodies and concanavalin A (Con A), and by 16 h had lost their binding sites for fucose binding protein (FBP) as well. That this reduction in antigenicity was due to shedding of surface antigens was suggested by the observation that the reduced ability of these parasites to bind anti-parasite antibodies coincided closely with the loss of 125I-labelled surface proteins. Furthermore unlike 6 day schistosomula which had developed wholly in vivo, 6-day-cultured parasites recovered after 30 min in vivo failed to bind anti-host antibodies suggesting that in these organisms parasite antigens were not masked by host molecules. These data argue that surface antigen shedding may explain the reduced surface antigenicity of schistosomula developing in vivo. While this surface modulation apparently occurs independently of host antigen uptake, it is dependent upon an as yet unidentified host factor.

Isolation and characterization of Z-DNA binding proteins from wheat germ

The preparation of a heterogeneous non-histone protein extract from wheat germ utilizing Br-poly(dG-dC).poly(dG-dC) (Z-DNA) affinity chromatography is described. The binding characteristics of antibodies against Z-DNA are used as a model system to define important criteria that the DNA binding behavior of a Z-DNA binding protein should display. We show that the wheat germ extract contains DNA binding proteins specific for left-handed Z-DNA by these criteria. The affinity of the proteins measured by competition experiments was approximately 10(5) greater for Br-poly(dG-dC).poly(dG-dC) (Z-DNA) than for poly(dG-dC).poly(dG-dC) (B-DNA). The affinity of the proteins for plasmid DNA increases with increasing negative superhelicity which is known to stabilize Z-DNA. The proteins are shown to compete with Z-DNA antibodies for binding to supercoiled plasmids. Finally, the affinity for two plasmids at a given superhelical density is greater for the plasmid containing an insert known to form Z-DNA than for a plasmid without the insert. The proteins exhibit a 2-3-fold greater affinity for stretches of (dC-dA)n.(dT-dG)n over stretches of (dG-dC)n.(dG-dC)n when both sequences are induced to form Z-DNA by supercoiling.

Binding of anti-galactosyl antibodies to galactosylated liposomes

The binding characteristics of rabbit anti-galactosyl antibodies elicited through galactosylated liposomes were studied. The association constants (13 × 10 5 M −1 and 21 × 10 5 M −1 of Fabμ and Fab fragments with the galactosylated liposomes were determined by measuring the binding of radioiodinated antibodies. The effect of free ligand (methyl-β- d-galactopyranoside) and the ligand coupled to the liposomal surface (galactosylated liposomes) on the structure of antibodies was investigated using circular dichroism (CD) measurements. Changes were observed in both the secondary and tertiary structure of antibodies upon binding to galactosylated liposomes. In contrast, the free ligand induced alterations only in the near-UV region of the CD spectra, indicating a change in tertiary structure only.

Identification and functional characterization of two distinct epitopes on the human T cell surface protein Tp50.

The structural and functional domains of Tp50, the human T lymphocyte surface protein associated with the E rosette receptor, were probed with the use of two murine monoclonal antibodies. Lysostripping, immune precipitation, and competitive binding experiments demonstrated that antibodies 9.6 and 35.1 bind to Tp50 epitopes in close proximity. In functional studies, both antibodies caused a similar degree of antigenic modulation, inhibited T cell proliferative responses, and inhibited cytotoxic T lymphocyte function without affecting cells that mediate antibody-dependent cell-mediated cytotoxicity. The antibodies were strikingly different, however, in that antibody 9.6 inhibited E rosette formation and natural killer cell-mediated lysis, whereas antibody 35.1 did not. These results could not be ascribed to differences in antibody class or binding characteristics, because Scatchard analysis demonstrated that these two IgG2a antibodies have comparable avidity and that T cells bind each antibody in equivalent amounts. The differential binding of antibodies 9.6 and 35.1 to T cells from nonhuman primates further supports the interpretation that the differences between the antibodies in their effects on E rosette formation and natural killer function stem from the fact that they bind to distinct epitopes of Tp50. The implications of these findings for understanding the functions of Tp50 molecules are discussed.

Insulin, pancreatic polypeptide, and glucagon antibodies in insulin-dependent diabetes mellitus.

To assess the possible value of the use of high-purity pork insulin (HPPI) in the United States, the serum insulin (I), pancreatic polypeptide (PP), glucagon (G), and somatostatin (SRIF) antibody binding characteristics have been determined in 90 conventional insulin-treated diabetic subjects and related to their degree of metabolic control, as assessed by glycosylated hemoglobin (HbA1) concentration. All diabetic subjects had antibodies to insulin, but there was no relationship between any of the antibody binding characteristics and HbA1 level: 47% possessed PP antibodies; mean +/- SEM HbA1 in these patients was 14.5 +/- 0.3%, identical to those without PP antibodies (14.5 +/- 0.4%); 10% had G binding antibodies with HbA1 levels of 14.6 +/- 0.8%, similar to those without G antibodies. No subject possessed SRIF antibodies. This lack of correlation between antibody characteristics and metabolic control makes it unlikely that, in the majority of patients, treatment with a less immunogenic insulin (HPPI) versus conventional insulin will result in improved diabetic control.

Binding characteristics of antibodies to the β-subunit of ovine luteinizing hormone (oLHβ) with human and rhesus gonadotropins

Binding characteristics of antibodies to the β-subunit of ovine luteinizing hormone (oLHβ) with human and rhesus gonadotropins

The relationship between circulating free and bound insulin, insulin antibodies, insulin dosage and diabetic control in insulin treated diabetics.

Free and bound insulin concentrations, blood glucose and anti-insulin antibody binding characteristics have been determined in 100 insulin treated diabetics; medium serum free insulin was 11 mU/l when fasting and rose to 30 mU/l after the mid-day meal. Significant correlation between blood glucose and serum free (but not bound) insulin was found. No relationship between insulin antibodies and daily insulin dose or diabetic control were found, nor a relationship between free insulin and antibody characteristics.

C-peptide secretion and insulin antibodies as determinants of stability in diabetes mellitus

The relative significance of residual β-cell secretory activity and human insulin antibodies in determining diabetic stability has been examined in 35 diabetic subjects. The response of plasma C-peptide immunoreactivity following 50 g oral glucose has been used as an index of β-cell function. Glucose-stimulated C-peptide secretion was observed in 58% of stable diabetics, but in no labile diabetics. When present, C-peptide secretion following a glucose load in diabetics was of smaller amplitude and slower in onset, but more prolonged than in normal subjects. In secretors, stability of diabetes was significantly correlated with the magnitude of the C-peptide response. As a group, labile diabetics had lower insulin antibody levels than stable patients, but stability and antibody levels were not correlated in individual patients. In non-secretors there was no difference in antibody levels between the stable and labile groups. Neither the equilibrium binding affinities nor the dissociation rate constants differed significantly for antibodies from stable and labile diabetics. Thus stability of diabetes depends upon residual β-cell secretory activity, but not on the concentration or binding characteristics of insulin antibodies.

Clonal Nature of the Immune Response to Phosphorylcholine

Abstract The fine specificity of anti-phosphorylcholine antibodies was studied in different rodent species and 17 different inbred mouse strains. Measurements of the relative affinity and specificity of the antibodies were derived from a hemolytic plaque-inhibition assay with phosphorylcholine and two of its analogues, glycerophosphorylcholine and choline, as inhibitors. Within a given species, inhibition of plaque-forming cells was accomplished within a narrow range of ligand concentration and little individual variation was seen. Moreover, no changes in either the slopes or relative position of the inhibition curves occurred at different times after immunization. These findings suggest that a restriction in heterogeneity of the anti-phosphorylcholine antibodies exists in each of the rodent species. However, striking species-specific differences were observed in the binding characteristics of antibodies from different species of rodents.