Antibody Reactivity Patterns Research Articles

Autoantibodies Against Arterial Antigens: Characterization by Elisa and Immunoblot Analysis in the Spontaneously Hypertensive Rat

The presence of autoantibodies directed against arterial antigens in serum samples from spontaneously hypertensive rats and related controls that included Wistar-Kyoto and Sprague-dawley rats were assayed by enzyme-linked immunosorbent assay and immunoblotting technique. Circulating immunoglobulin G antibodies reactive against arterial antigen, as measured by enzyme-linked immunosorbent assay, could be detected in serum samples of 26 of 30 spontaneously hypertensive rats (87%) and 8 of 30 (27%) Wistar kyoto rats. These antibodies (Abs) were not detectable either by enzyme-linked immunosorbent assay or immunoblotting in sera from Sprague-dawley rats. The arterial antigen-reactive antibody was antigen specific, because the binding reactivity was absorbed by arterial antigen but not by fibroblasts or peripheral blood mononuclear cells. Immunoglobulin G arterial antigen-reactive antibody was significantly higher in adult spontaneously hypertensive rats with established hypertension, compared with young prehypertensive rats or normotensive wistar kyoto rats. Immunoblotting of spontaneously hypertensive rats sera revealed reactivity of arterial antigen-reactive antibody against arterial antigen ranging in size from 20 to 97 kDa. Sera from Wistar kyoto rats recognized arterial antigen ranging in size from 40 to 90 kDa. A significant correlation (p < 0.004) was found between adult spontaneously hypertensive rats with established hypertension and the presence of arterial antigen-reactive antibody reactivity against arterial antigen of 20, 69 and 97 kDa. Antibody directed against a 20 kDa arterial antigen was detected in both young prehypertensive rats and adult rats with established hypertension but not in Wistar kyoto or Sprague-dawley rats. Antibodies directed against both 69 and 97 kDa arterial antigens were detected only in spontaneously hypertensive rats sera. These data show that the pattern of arterial antigen-reactive antibody reactivity in sera of hypertensive rats in heterogeneous, and suggest that arterial antigen-reactive antibody directed against few arterial antigens may be involved in the pathogenesis of hypertension in the spontaneously hypertensive rat.

Detection of autoantibodies directed against human hepatic endoplasmic reticulum in sera from patients with halothane‐associated hepatitis.

1. Previous studies have demonstrated the presence of antibodies to trifluoroacetylated hepatic proteins (TFA-proteins) in sera from patients with the severe form of halothane-associated hepatitis (halothane hepatitis). The TFA-proteins are produced via cytochrome P450-mediated metabolism of halothane to the reactive species TFA-chloride. 2. To investigate the presence of autoantibodies (which recognize various non-TFA-modified human hepatic polypeptides) in patients with halothane hepatitis immunoblotting experiments were performed using microsomal fractions prepared freshly from livers of five different (halothane-free) tissue donors. Blots were developed using 15 well-characterised sera from patients with halothane hepatitis. 3. Autoantibodies to human hepatic polypeptides were detected in most, but not all, of the patients' sera. The pattern of antibody reactivity varied markedly between sera. Although no common pattern of antibody recognition was observed, polypeptides of molecular mass between 60 and 80 kDa were the predominant targets. A similar protein recognition pattern was seen when each positive serum was tested against the five individual human liver samples. 4. Such autoantibodies were not detected in sera from 16 normal human blood donors, but were detected in three of six sera from patients exposed to halothane without developing hepatitis. 5. The autoantibodies are thought to arise in patients exposed to halothane as a consequence of a halothane-induced immune response to chemically-modified proteins. Such antibodies could contribute to the complex pathological processes involved in halothane hepatitis.

Antibody responses of Porphyromonas gingivalis infected gingivitis and periodontitis subjects

Porphyromonas gingivalis demonstrates a strong association with adult periodontitis although some individuals with the infection do not experience attachment loss. Therefore differences in the immune response to this organism may be of importance to the outcome of the disease. The aim of this study was to determine whether P. gingivalis positive subjects with and without periodontal breakdown, reacted differently to P. gingivalis antigens as assessed by the pattern of serum antibody reactivity. Two highly defined groups of subjects were chosen for this study. Both demonstrated P. gingivalis in their plaque and both had responded to P. gingivalis as shown by the presence of serum antibodies. The two groups differed only in their apparent clinical susceptibility to periodontal breakdown. Western blots of P. gingivalis membrane antigens were probed with sera from the two groups to determine their reactivity to specific antigens. Analysis of the immunoblots showed that there were no differences in either the total numbers of bands, or bands recognized by the majority of subjects in the gingivitis and adult periodontitis groups. There were however, four bands recognized by the majority of the gingivitis group and not by the majority of the adult periodontitis group, there being a significant difference (P = 0.03) in the recognition of the 91.4-kDa antigen band. A further five antigens of lower molecular weight were seen by the majority of the adult periodontitis group and not by the majority of the gingivitis group. When sera were tested against purified P. gingivalis LPS, the results indicated that the five antigens seen by the majority of the adult periodontitis group had molecular weights which were in the range exhibited by the LPS antigens. These results suggest that gingivitis and adult periodontitis subjects with P. gingivalis infection, may recognize different P. gingivalis antigens.

Nucleotide and deduced amino acid sequence of the structural protein genes of Japanese encephalitis viruses from different geographical locations

Strain variation among Japanese encephalitis (JE) virus isolates has been previously demonstrated by many workers using different methods. We report the nucleotide sequence of the 5' non-coding region and the nucleotide and deduced amino acid sequences of structural protein genes for eight wild-type JE virus strain isolated from different Asian countries (Vietnam, Nepal, Indonesia, Thailand, India, Japan and China). We compare these with five other published wild-type JE virus strains isolated from Japan and the mainland of China. No strain variation could be related to geographical location on the basis of the wild-type JE virus strains studied. The viruses differed from each other by between 21 (0.9%) and 111 (4.6%) nucleotides and by between 3 (0.4%) and 31 (4.2%) amino acids over the 2434 nucleotides examined. The amino acid divergence of the envelope (E) protein gene of the viruses was 4.2% or less among the 13 strains compared. Thus there is little genetic divergence in the strains studied. However, four variable E protein amino acids (E-51, E-209, E-244 and E-366) were identified. Residue positions E-51, E-244 and E-366 are found in peptides with functional T helper cell epitopes in two other flaviviruses. Therefore, these amino acids may be important in defining the various immunotypes of JE virus identified by antibody reactivity patterns.

Interference of immunoglobulin G (IgG) antibodies in IgA antibody determinations of Chlamydia pneumoniae by microimmunofluorescence test

In the microimmunofluorescence test for measuring immunoglobulin A (IgA) antibodies against Chlamydia pneumoniae, removal of interfering IgG antibodies made IgA antibody reactivity patterns in 952 serum samples easier to interpret, prozone effects disappeared, and titers increased, especially in the sera with high IgG titers. IgA rheumatoid factors did not interfere in the assay.

Circulating cornea-specific antibodies in corneal disease and cornea transplantation.

In order to establish the significance of circulating cornea-specific antibodies, we determined the presence of anti-corneal antibodies in the serum of 100 patients with corneal disease and in 50 healthy controls, and subsequently followed the pattern of antibody reactivity in 46 patients who underwent corneal transplantation. An indirect immunofluorescence test on cryostat sections of rabbit corneas was used for screening. The reactivity against two known bovine corneal epithelial proteins was also tested: a 54-kD protein (BCP 54) and an 11-kD protein (BCP 11/24). No significant difference in the presence and specificity of anti-corneal antibodies was observed between the group of patients with corneal disease, taken as a whole, and the healthy controls. Patients with keratoconus or non-immunological graft failure, however, were significantly more often positive for anti-corneal antibodies. Neither the presence of antibodies prior to corneal transplantation nor their appearance post-transplantation had a predictive value for corneal graft survival.

Respiratory syncytial virus subgroup B dominance during one winter season between 1987 and 1992 in Vancouver, Canada

A subgroup analysis of 613 specimens submitted to the British Columbia's Children's Hospital from 1987 to 1992 revealed that subgroups A and B of respiratory syncytial virus (RSV) were both circulating in our community, with some predominance for subgroup A during the period from October 1987 to September 1988 (the 1987-88 season) (64%), 1990-91 (60%), and 1991-92 (62%). During 1989-90 subgroup A represented the majority of isolates (80%). Subgroup B predominated during only one season, 1988-89 (94%). No microheterogeneity within subgroups was apparent as judged by the monoclonal antibody reactivity pattern. More male than female children were affected overall, but no sex-related difference between subgroup infections could be detected (P = 0.28). The majority of patients were less than 1 year of age, and no significant association between age and subgroup was detected after stratifying for year (P = 0.64). This is, to our knowledge, the first comprehensive longitudinal RSV subgroup prevalence study from the Pacific Northwest and from Canada.

Transfer of humoral immunity against cytomegalovirus proteins following transplantation of T‐cell‐depleted allogeneic bone marrow from seropositive donors

Previous work by Grob et al. [Lancet i: 774, 1987] has demonstrated that allogeneic, T-cell-depleted bone marrow transplant recipients have a better prognosis for reactivated cytomegalovirus (CMV) infection if their donor is also immune. It was proposed that adoptively transferred humoral immunity was responsible for the protective effect of active infection. Immunoblot analysis using purified virions was used here to examine pre- and posttransplant antibody responses of seropositive recipients who had undergone active viral infection after transplantation. Immunoblots were assessed for the numbers of polypeptides recognised and reactivity against individual polypeptides. Immunoblots were also scanned by quantitative densitometry, and the intensity of antibody responses against total viral protein and individual polypeptides was determined. Sera from recipients with immune donors exhibited a secondary-type immune response in terms of both intensity and polypeptide specific pattern of antibody reactivity, compared with those recipients with nonimmune donors. In particular, recipients with immune donors appeared to show a greater reactivity against a protein of M(r) 55,000; this may represent the envelope glycoprotein gB, which is a major target for neutralising antibodies, and might also be utilised for preparing an effective vaccine for CMV.

Tissue Printing as a Tool for Observing Immunological and Protein Profiles in Young and Mature Celery Petioles.

Tissue printing onto membranes such as nitrocellulose is a technique employed to study the localization of proteins, nucleic acids, and soluble metabolites from freshly cut tissue slices. We probed tissue prints of young and mature celery (Apium graveolens) petioles with antibodies raised against two proteins, spinach ribulose-1,5-bisphosphate carboxylase and tomato fruit catalase. The purposes of this study were to determine if these proteins are developmentally regulated and to determine if the patterns and intensities of cross-reactivity of antibodies on tissue blots corresponded only to the presence of specific epitopes or was related to the amount of protein present in any given area on the tissue prints. Different and distinct cross-reactivity patterns were observed with each of the two antibodies used. Tissue prints from young and mature tissues also showed differences in antibody cross-reactivity. Comparison of Coomassie blue staining patterns with antibody reactivity patterns showed that there is little relationship between tissue protein concentration and antibody reactivity.

Distinct Pattern of Antibody Reactivity with Oligomeric or Polymeric Forms of the Capsular Polysaccharide of Haemophilus influenzae Type b

Distinct Pattern of Antibody Reactivity with Oligomeric or Polymeric Forms of the Capsular Polysaccharide of <i>Haemophilus influenzae</i> Type b

Immunohistochemical localization of matrix proteins in the femoral joint cartilage of growing commercial pigs.

The immunocytochemical localization of several matrix macromolecules, including collagen type II and proteoglycans, in the distal femoral articular-epiphyseal cartilage complex of 15 commercial pigs between the age of 6 and 18 weeks was studied. Early osteochondrotic lesions, i.e., chondronecrosis in the resting region of the growth cartilage, as well as extensions of necrotic cartilage into the subchondral bone, were present in all animals, except those 6 weeks old. A battery of antibodies were used for identification of macromolecules in the matrix at different stages of the disease. Chondrocyte involvement in the process could be studied by identifying the sequence of alterations in matrix macromolecules as the lesion developed. The immunostaining for aggrecan (large aggregating proteoglycans), cartilage oligomeric matrix protein, fibronectin, collagen type II, fibromodulin, and biglycan was more prominent in the areas of chondronecrosis, extending into the subchondral bone, than in the normal resting region. This altered pattern of matrix macromolecules resembled that of the matrix of the proliferative chondrocytes and suggests that the chondrocyte maturation had stopped in the proliferative zone. The matrix in the areas of chondronecrosis in the resting region resembled that in the normal resting region. Thus the chondronecrosis appears to have preceded alterations of the matrix composition. The antibody reactivity pattern was, however, altered in the matrix of the clustered chondrocytes in areas of chondronecrosis. Staining in these regions suggested a more prominent appearance of fibronectin and collagen type II than in the normal matrix of the resting region. These changes are suggestive of attempt to repair.(ABSTRACT TRUNCATED AT 250 WORDS)

The human repertoire of antibody specificities against Thomsen-Friedenreich and Tn-carcinoma-associated antigens as defined by human monoclonal antibodies.

Human monoclonal antibodies specific for tumour-associated Thomsen-Friedenreich (TF) [Gal(beta 1-3)GalNAc(alpha)-O-] and Tn [GalNAc(alpha)-O-] glycoproteins were prepared using peripheral blood lymphocytes from healthy blood donors. The B lymphocytes were either directly transformed with Epstein-Barr virus (EBV) or transformed after an in vitro stimulation period with synthetic glycoproteins. The EBV-transformed lymphocytes were subsequently fused with a mouse-human heteromyeloma to secure antibody production and stability. IgM antibodies exhibiting different patterns of specificity for synthetic TF and Tn antigens were obtained, including antibodies specific for the alpha and beta forms of different Gal(beta 1-3)GalNAc-O- and GalNAc-O- conjugates and antibodies agglutinating neuraminidase-treated erythrocytes. Several of the human monoclonal antibodies showed an increased binding to cultured carcinoma cells as compared to melanoma cells. This straightforward approach for the production of human monoclonal antibodies demonstrates the possibility of investigating the reactivity pattern of tumour-binding antibodies from peripheral blood lymphocytes. The binding patterns of these monoclonal antibodies show that healthy donors carry different fine specificities against synthetic TF/Tn antigens and that these antibodies react with different tumour cells.

Intestinal brush-border-associated enzymes: co-ordinated expression in colorectal cancer.

The brush border of normal small-intestine epithelial cells is rich in enzymes that are involved in the digestive process. Such molecules can be used as markers to analyze cell lineages and differentiation properties of colorectal cancers. Monoclonal antibodies detecting dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase, alkaline phosphatase, maltase-glucoamylase and lactase have been used to analyze the phenotype of colorectal cancers, adjacent mucosa and histologically normal distant mucosa. The avidin-biotin peroxidase complex method was used. Expression of dipeptidyl peptidase-IV, aminopeptidase N, sucrase-isomaltase and alkaline phosphatase was common in non-neoplastic mucosa adjacent to, and distant from, the tumor; in contrast, endopeptidase F, maltase-glucoamylase and lactase were rarely expressed in normal distant mucosa and more frequently expressed in mucosa adjacent to the tumor. Dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase and alkaline phosphatase were frequently expressed in colorectal cancers, whereas maltase-glucoamylase and lactase were rarely expressed. Two general patterns of antibody reactivity were observed: diffuse cytoplasmic and apical; apical reactivity was generally associated with more differentiated tumors. A logistic predictive regression model indicated that enzyme expression in colorectal cancers followed a coordinate pattern, but was unrelated to the location of the tumor, Dukes stage or differentiation grade. In conclusion, expression of brush-border-associated enzymes occurs frequently in colorectal cancers and is regulated in a co-ordinated manner. These markers can be used for the phenotypic sub-classification of colorectal cancers.

Monoclonal antibodies raised against pre-migratory neural crest reveal population heterogeneity during crest development

Abstract In order to address the problem of when heterogeneity arises within premigratory and early migratory neural crest cell populations, mouse monoclonal antibodies were raised against quail premigratory neural crest. Due to the limited availability of immunogen an intrasplenic route for immunization was used. Three monoclonal antibodies (referred to as LH2D4, LH5D3 and LH6C2) were subsequently isolated which recognized subpopulations in 24 h cultures of both quail and chick mesencephalic and trunk neural crest in immunocytochemical studies. Subsequent investigations using a range of six antibodies, including LH2D4, LH5D3 and LH6C2, showed that population heterogeneity (which was not cell cycle related) could be detected as early as 15 h following mesencephalic crest explantation, a stage at which all the neural crest cells were morphologically identical. However, premigratory neural crest from the same axial level of origin was homogeneous, as judged by immunoreactivity patterns with these antibodies. Significant differences were found in the proportion of immunoreactive cells between populations of mesencephalic and trunk neural crest cultures. Double immunofluorescence studies revealed the existence of at least four separate cell populations within individual crest cultures, each identified by their unique antibody reactivity pattern, thus providing some insight into the underlying complexity of subpopulation composition within the neural crest. Immunocytochemical studies on quail embryos from stages 7–22 showed that the epitopes detected by LH2D4, LH5D3 and LH6C2 were not necessarily confined to the neural crest or to cells of crest derivation. All three epitopes displayed a spatiotemporal regulation in their expression during early avian ontogeny. Since the differential epitope expression described in this investigation was detectable as early as 15 h after premigratory neural crest explantation, took place in vitro in the absence of any other cell type and changed progressively with time, we conclude that a certain degree of population heterogeneity can be generated very early in neural crest ontogeny and independently of the tissue interactions that normally ensue in vivo.

Identification and localization of a limited number of predominant conformation-independent antibody epitopes of Theiler's murine encephalomyelitus virus.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease in mice is a well established animal model for human multiple sclerosis (MS). Identification of pathogenic epitopes may be helpful in understanding the pathogenesis of this immune-mediated disease. In order to analyze the viral epitopes, we have generated approx. 150 recombinant lambda gt11 clones expressing various capsid areas of TMEV. Six predominant areas, ranging from 13-26 amino acid residues, (3 in VP1, 2 in VP2 and 1 in VP3) are readily recognized by conformation-independent antibodies from virus-infected mice. These areas have been designated as A-1A (VP1 13-27th residues), A-1B (VP1 145-167), A-1C (VP1 251-276), A-2A (VP2 2-14), A-2B (VP2 165-179), and A-3A (tentatively VP3 24-43). Antibodies from TMEV-infected susceptible SJL/J mice strongly react with A-1B, A-2A and A-2B, in contrast to antibodies from resistant BALB/c mice which mainly recognize A-1A and A-2A. Interestingly, the reactivity pattern of antibodies from TMEV-infected mice are somewhat different from that of antibodies from TMEV-immunized mice. Although the majority of antibodies in TMEV-infected mice recognizes conformation-dependent epitopes, the differential recognition of the conformation-independent antibody epitopes by susceptible mice may play a role in TMEV-induced demyelination.

Different patterns of glycolipid antibody reactivity: lower motor neuron syndromes vs. immunization

High titers of serum antibodies against G M1 ganglioside occur frequently in patients with lower motor neuron (LMN) syndromes. We compared the specificities of the antiganglioside antibody reactivities in LMN patients with those arising after immunization of Lewis rats with several ganglioside containing preparations including purified G M1, human central nervous system (CNS) grey matter and white matter. Serums with high titers of anti-G M1 antibodies from patients with LMN syndromes usually showed limited cross-reactivity to other glycolipids but often bound to a Gal(β1-)GalNAc-containing neoglycoprotein. In contrast, serums with anti-G M1 antibody arising after immunization showed broad cross-reactivity with other glycolipids but did not bind to the neoglycoprotein. We conclude that the serum patterns of antiganglioside antibody reactivity secondary to immunization with gangliosides and CNS components are different from the natural autoantibodies found in LMN patients. The antiganglioside antibodies seen in LMN patients are unlikely to be a result of autoreactivity to gangliosides after nervous tissue damage.

Potyviruses, monoclonal antibodies, and antigenic sites.

Virus-specific and cross-reactive monoclonal antibodies have been produced to at least 19 different aphid-transmitted potyviruses. This report summarizes the development of these monoclonal antibodies as well as presents information on the delineation of the virus-specific and group-common epitopes defined by these monoclonal antibodies. Virus-specific and group-common antigenic determinants were mapped by a variety of techniques, including analysis of antigen: antibody reactivity patterns, determination of N-terminal vs. trypsin-resistant core peptide-specificity, immunoanalysis of overlapping synthetic peptides, and immunoanalysis of bacterially expressed coat-protein gene products. Of those monoclonal antibodies that have been examined, monoclonal antibody-defined virus-specific epitopes are virion surface-located within the 30+ amino acid amino terminus, whereas the group-common epitopes are found in the trypsin-resistant core protein not usually located on the virion surface, as has been shown previously with certain polyclonal antibodies. New information is presented on the analysis of bean yellow mosaic virus amino terminal epitopes as well as on the identification of amino terminal antigenic determinants shared between strains of bean yellow mosaic virus and pepper mottle virus. A recommendation on the evaluation and use of a panel of potyvirus broad-spectrum reacting monoclonals as reference monoclonal antibodies for the detection and classification of aphid-transmitted potyviruses is also presented.

Unusual pattern of antibodies to human T-cell leukemia virus type-I in family members of adult T-cell leukemia patients

Detection methods for the human T-cell leukemia virus type-I (HTLV-I) for blood screening and diagnosis generally rely on antibody tests that use the structural proteins of HTLV-I as antigen. We have found an unusual pattern of antibody reactivity among people who are at high risk of HTLV infection due to being a family member of an adult T-cell leukemia (ATL) patient: a specific antibody reaction exclusively directed to the HTLV regulatory protein tax, and not to the HTLV-I structural proteins. Sera from 7 of 82 (8.5%) structural antibody-undetectable family members of ATL patients had the anti-tax reactivity. Two seroconverters were observed. One seroconverter a healthy resident of Miyazaki, tested negative for structural antibody, but positive for tax antibody. Two years later she tested positive for both. The other seroconverter, an Israeli hemophiliac, tested negative for both antibodies, but converted to tax antibody-positive/structural antibody-negative. The HTLV-I tax-only antibody profile was also observed in sera sets from two other populations at risk for HTLV infection, human immunodeficiency virus-1-infected patients at the Bronx-Lebanon Hospital in New York and Israeli hemophiliacs. DNA samples from lymphocytes of four individuals with antibody reactivity only to HTLV-I tax were tested in polymerase chain reaction experiments; no HTLV-I or -II DNA was detected.

Unusual Pattern of Antibodies to Human T-Cell Leukemia Virus Type-I in Family Members of Adult T-Cell Leukemia Patients

Abstract Detection methods for the human T-cell leukemia virus type-I (HTLV-I) for blood screening and diagnosis generally rely on antibody tests that use the structural proteins of HTLV-I as antigen. We have found an unusual pattern of antibody reactivity among people who are at high risk of HTLV infection due to being a family member of an adult T-cell leukemia (ATL) patient: a specific antibody reaction exclusively directed to the HTLV regulatory protein tax, and not to the HTLV-I structural proteins. Sera from 7 of 82 (8.5%) structural antibody- undetectable family members of ATL patients had the anti-tax reactivity. Two seroconverters were observed. One seroconverter a healthy resident of Miyazaki, tested negative for structural antibody, but positive for tax antibody. Two years later she tested positive for both. The other seroconverter, an Israeli hemophiliac, tested negative for both antibodies, but converted to tax antibody-positive/structural antibody-negative. The HTLV-I tax-only antibody profile was also observed in sera sets from two other populations at risk for HTLV infection, human immunodeficiency virus-1-infected patients at the Bronx-Lebanon Hospital in New York and Israeli hemophiliacs. DNA samples from lymphocytes of four individuals with antibody reactivity only to HTLV-I tax were tested in polymerase chain reaction experiments; no HTLV-I or -II DNA was detected.

Distinct pattern of antibody reactivity with oligomeric or polymeric forms of the capsular polysaccharide of Haemophilus influenzae type b

The chain length of oligosaccharides required for antibody binding has been studied by using the capsular polysaccharide from Haemophilus influenzae type b or oligosaccharides derived from it. The concentration of competing antigens required to achieve a 50% inhibition of antibody binding by human polyclonal antisera in an in vitro competition enzyme-linked immunosorbent assay decreased progressively from greater than 10(-3) to 5 x 10(-7) M as the inhibiting saccharide chain length increased from 1 to 262 repeat units. Even small oligosaccharides (one or two repeat units) are potentially capable of competing to a significant level if a high enough concentration of saccharides is used. A similar pattern of reactivity was seen with a monoclonal anti-polyribosyl ribitol phosphate antibody, suggesting that the differences in the avidity of the antibody subpopulations in the polyclonal antisera do not contribute to the binding patterns observed. The binding reaction was specific as evaluated with pneumococcal saccharides. Furthermore, an oligosaccharide-protein conjugate binds antibody better than the free oligosaccharides do. Such a difference in binding was not observed between the polysaccharide and a polysaccharide-protein conjugate. Overall, the data suggest that identical epitopes are expressed by oligomeric and polymeric forms of the antigen and that a particularly more stable conformation in polysaccharides is preferred by antibodies. Covalent coupling of oligomers to protein increases the expression of stable conformation of epitopes. The data further suggest that this kind of antigenic analysis may be important for the design and synthesis of glycoconjugate vaccines.