Studies of a monoclonal antibody to skeletal keratan sulphate. Importance of antibody valency.

A mouse monoclonal antibody (AN9P1) to keratan sulphate is described. In a competitive-inhibition solution-phase radioimmunoassay employing 125I-labelled intact proteoglycan, it reacts preferentially with keratan sulphate bound to the core protein of adult human articular-cartilage proteoglycan and to a much lesser degree with keratan sulphate purified from this proteoglycan. Proteolytic cleavage of the proteoglycan by pepsin and trypsin has little effect on antibody binding, but treatment with papain decreases binding considerably and more than does treatment with keratanase. An even greater decrease in binding is observed after treatment with alkaline borohydride. A comparison of binding of antibody AN9P1 with that of another previously described monoclonal antibody, 1/20/5-D-4, to keratan sulphate [Caterson, Christner & Baker (1983) J. Biol. Chem. 258, 8848-8854] revealed similar binding characteristics, both showing much diminished binding after papain digestion of proteoglycan and even less with purified skeletal keratan sulphate. Removal of the Fc piece of antibody AN9P1 had no significant effect on the differential binding of divalent F(ab')2 fragment to proteoglycan, to papain-digested proteoglycan and to keratan sulphate, although there was a small decrease in binding to papain-digested proteoglycan. Conversion of the antibody into univalent Fab fragment with removal of the Fc piece resulted in diminished binding to proteoglycan, compared with that observed with IgG, and in enhanced binding to free keratan sulphate and to papain-digested proteoglycan. These results suggest that close proximity of keratan sulphate chains on the core protein of proteoglycans favours preferential reactivity of bivalent antibody with these species through cross-bridging of chains by antibody. Conversely, much decreased binding to keratan sulphate on proteoglycan core-protein fragments and to free keratan sulphate results from a lack of close proximity of keratan sulphate. By using univalent Fab fragment in these assays these differences in binding are minimized by preventing cross-bridging and thereby enhancing detection of smaller fragments without sacrificing too much sensitivity of detection of larger proteoglycan species. The persistent preferential binding of Fab fragment to proteoglycan is probably in part the result of the increased epitope density in the intact molecule compared with keratan sulphate in a more disperse form.     

Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan

Monoclonal antibodies were raised against proteoglycan core protein isolated after chondroitinase ABC digestion of human articular cartilage proteoglycan monomer. Characterization of one of the monoclonal antibodies (1/20/5-D-4) indicated that it specifically recognized an antigenic determinant in the polysaccharide structure of both corneal and skeletal keratan sulfate. Enzyme immunoassay analyses indicated that the mouse monoclonal IgG1 recognized keratan sulfate in native proteoglycan aggregate and proteoglycan monomer preparations isolated from hyaline cartilages of a wide variety of animal species (human, monkey, cow, sheep, chicken, and shark cartilage). The 1/20/5-D-4 monoclonal antibody did not recognize antigenic determinants on proteoglycan isolated from Swarm rat chondrosarcoma. This finding is consistent with several biochemical analyses showing the absence of keratan sulfate in proteoglycan synthesised by this tissue. A variety of substructures isolated after selective cleavage of bovine nasal cartilage proteoglycan (Heineg?rd, D., and Axelsson, J. (1977) J. Biol. Chem. 252, 1971-1979) were used as competing antigens in radioimmunoassays to characterize the specificity of the 1/20/5-D-4 immunoglobulin. Substructures derived from the keratan sulfate attachment region of the proteoglycan (keratan sulfate peptides) showed the strongest inhibition. Both corneal and skeletal keratan sulfate peptides as competing antigens in radioimmunoassays showed similar inhibition when compared on the basis of their glucosamine content. Therefore, the 1/20/5-D-4 monoclonal antibody appears to recognize a common determinant in their polysaccharide moieties. Chemical desulfation of the keratan sulfate reduced the antigenicity of the glycosaminoglycan. The antibody did not recognize determinants present in dermatan sulfate, heparin, heparin sulfate, or hyaluronic acid.     

Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate

We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan sulfate. Keratan sulfate-negative chondrocytes were shown to come from the upper cartilage layers. There was therefore a direct correlation between biochemical heterogeneity of cartilage matrix and heterogeneity within the chondrocyte population. During growth in monolayer culture, superficial chondrocytes began to synthesize keratan sulfate, but the cells could still be distinguished from cultures of deep or unfractionated chondrocytes by their reduced substrate adhesiveness and tendency to remain rounded.     

Monoclonal antibodies specific for keratan sulfate detect epithelial-associated carbohydrates.

Monoclonal antibodies that specifically recognize epitopes on keratan sulfate glycosaminoglycans were used in this study to identify carbohydrate epitopes associated with many, but not all, types of epithelial cells. Immunoreactive cells included: keratinocytes, sebaceous gland cells, eccrine sweat gland duct cells, salivary gland excretory duct cells, colon adenocarcinoma cells, embryonic chick lung epithelial cells, embryonic chick mesonephric and metanephric kidney epithelial cells, and selected embryonic chick neural tube cells. Depending upon the type of epithelium, epitopes were located either within the cytoplasm or were located on cell surfaces. These epitopes were shared by cells from both human and chick tissues, indicating the absence of species specificity. Not all anti-keratan sulfate antibodies were equally effective in identifying epithelial-associated epitopes. One of the seven antibodies employed in this study failed to detect epitopes in almost all epithelial tissues studied. Of the remaining six antibodies, three were more effective than the others in recognizing epithelial-associated epitopes. These data indicate that carbohydrates that are typically associated with extracellular matrix can also be associated with epithelial cells, but in a form that is not necessarily related to extracellular matrix. These antibodies should prove to be useful in studies of the development of epithelial cells and tissues.     

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Active site structure and antigen binding properties of idiotypically cross-reactive anti-fluorescein monoclonal antibodies

This report includes complete VH and V kappa nucleotide and deduced amino acid sequences of idiotypically cross-reactive monoclonal anti-fluorescein antibodies that differed greater than 10(5)-fold in affinity. High affinity monoclonal antibody 4-4-20 and intermediate affinity antibodies 10-25, 5-14, 9-40, 12-40, and 3-24 utilized greater than or equal to 90% homologous VHIIIC germ-line genes. Extensive D segment length and sequence variability were observed; however, compensatory germ-line JH4 (4-4-20 and 3-24) or JH3 (10-25, 5-14, 9-40, and 12-40) sequence lengths resulted in H chain CDR3 + FR4 to be a constant 18 amino acids. In addition, each antibody and low affinity 3-13 rearranged greater than or equal to 96% homologous V kappa II genes to J kappa 1, except for 10-25 (J kappa 5) and 3-13 (J kappa 4). Resolved crystal structure of complexed fluorescein and 4-4-20 Fab fragments revealed residues HisL27d, TyrL32, ArgL34, SerL91, TrpL96, and TrpH33 acted as hapten contact residues. Antibodies 5-14, 9-40, 12-40, and 3-24 primary structures possessed identical contact residues as 4-4-20 except for the substitution of HisL34 for ArgL34. Thus, ArgL34 was implicated in the increased affinity of monoclonal antibody 4-4-20. Finally, it was difficult to correlate extensive H chain CDR3 residue heterogeneity directly with fluorescein binding and idiotypy.     

Antigenic binding sites of monoclonal antibodies specific for simian virus 40 large T antigen.

We isolated 16 new monoclonal antibodies that recognize large T antigen of simian virus 40 and mapped the epitopes to three distinct regions of the large T antigen. Also, 3 of the 16 recognized the large T antigen of the human papovavirus BKV.     

Anti-bilirubin monoclonal antibody. III. Preparation and properties of monoclonal antibodies to unconjugated bilirubin-IX alpha

Anti-bilirubin-IX alpha monoclonal antibodies exclusively specific for unconjugated bilirubin-IX alpha are prepared and characterized. Using modified MBS (metamaleimidobenzoyl-N-hydroxysuccinimide ester) method, bilirubin-IX alpha was covalently coupled to bovine serum albumin (BSA) retaining its intramolecular hydrogen bonds as well as three-dimensional structure. Somatic cell fusion was performed between murine spleen cells immunized with the bilirubin-IX alpha-BSA and murine myeloma P3-X63-Ag8-U1 cells. After screening assay, 58 clones were identified which were producing antibodies not to albumin nor MBS, but to haptenic bilirubin-IX alpha. In inhibition analysis, the antibodies in the culture supernatant reacted only with bilirubin-IX alpha to a varying degree, but did not react with conjugated bilirubin-IX alpha, including bilirubin-IX alpha monoglucuronide, bilirubin-IX alpha diglucuronide, and ditauronic bilirubin-IX alpha.     

MZ15, a monoclonal antibody recognizing keratan sulphate, stains chick tendon

Summary Biochemical, morphometric and electron histochemical methods have failed to demonstrate the presence in tendon of keratan sulphate, a common component of connective tissue proteoglycans. Using a monoclonal antibody specific for keratan sulphate, a positive localization of this molecule was made in the gastrocnemius tendon of stage 44 chicken embryos both at the light and electron microscopical levels.     

Biosynthesis of keratan sulfate: purification and properties of a galactosyltransferase from bovine cornea.

A soluble galactosyltransferase was purified 22,000-fold from bovine cornea. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-acetyl-d-glucosamine, α- and β-glucosaminides, bovine cornea and nasal septum agalactokeratan, and to glycoproteins containing terminal nonreducing N-acetylglucosaminyl units. When N-acetyl-d-glucosamine served as acceptor, the product formed by the cornea transferase contained galactose glycosidically linked to carbon atom 4 of N-acetyl-d-glucosamine; the same glycosidic linkage was found in [¹⁴C]keratan preparations isolated from reaction mixtures where keratan containing terminal nonreducing N-acetylglucosaminyl units served as acceptor. The cornea enzyme exhibited a markedly lower K_m with keratan than with N-acetyl-d-glucosamine. The physical and kinetic properties of the cornea galactosyltransferase and of the milk A-protein (A-protein + α-lactalbumin = lactose synthase), including modulations of acceptor specificity by α-lactalbumin, were compared. The results of these studies strongly suggest that the two glycosyltransferases are similar, if not identical. Efforts to demonstrate the presence of other soluble galactosyltransferases in cornea were unsuccessful; no change in the ratios of products formed with several acceptors was observed at any stage of purification. It is suggested that in bovine tissues a single galactosyltransferase participates in the synthesis of both high and low molecular weight galactosides including the assembly of the repeating disaccharide [O-β-galactopyranosyl-(1 → 4)-N-acetylglucosamine] of cornea keratan sulfate.     

Antigenic mosaic of Methanogenium spp.: analysis with poly- and monoclonal antibody probes.

Eight well-characterized Methanogenium strains, including the six described type strains, were analyzed with poly- and monoclonal antibody probes to examine the antigenic mosaic of the genus. The pattern of cross-reactions showed that the mosaic is complex and varies with the strains; thus, these organisms have developed a considerable antigenic diversity, which is expressed in their envelopes. Every strain shared at least one determinant with at least one other strain, demonstrating the antigenic cohesiveness of the group. This finding, together with the fact that most strains displayed a distinctive antigenic fingerprint (notwithstanding the limited number of probes available), emphasizes the potential of antibodies for rapid identification of new isolates and for direct elucidation of Methanogenium strains in microbial mixtures.     

Monoclonal antibody immunocytochemistry: novel method extending usefulness of monoclonal antibodies for antigen visualization

We describe a novel procedure combining the multiple-site reactivity of polyclonal antibodies with the defined single epitope-specificity of monoclonal antibodies. The method is based on previous findings that IgG molecules often only react with tissue-bound antigens with one of their two antigen-combining sites; thus, the remaining site is free to bind subsequently added antigen. In the procedure devised, such (undenatured) antigen is subsequently detected by a specific monoclonal antibody and the reaction is finally revealed by immunogold-silver staining. Antibody subpopulations to contaminating antigens may well be present in the polyclonal antiserum and may well bind first to tissue and then to the corresponding contaminants in the crude antigen preparation applied as second layer. Such contaminants will, however, not react with the monoclonal antibody and will therefore not be immunocytochemically detected. The method has been evaluated with one antigen which cannot be detected by monoclonal antibodies in paraffin sections (glial fibrillar acidic protein) and with another antigen (human chorionic gonadotropin) which can only be detected by the monoclonal antibody when occurring in high concentrations. In both cases the procedure resulted in strong specific staining of the antigens with no background.     

Antigenic structure of hepatitis B surface antigen: identification of the "d" subtype determinant by chemical modification and use of monoclonal antibodies

Hepatitis B surface antigens (HBsAg) of both the adw and ayw subtypes were reductively methylated with formaldehyde in the presence of sodium cyanoborohydride. The effect on antigenicity was determined by radioimmunoassay with monoclonal antibodies specific for seven different antigenic determinants. The reaction was shown to eliminate specifically the "d" antigenic activity of HBsAg/adw and to have no effect on HBsAg/ayw. Moreover, the reaction had only a slight affect on HBsAg/adw at one of the "a" antigenic determinants. The sites of modification were determined and the extent of modification of each site was compared to the loss of "d" antigenic activity. These studies demonstrated that the loss of "d" activity was due to the modification of lysine 122 in HBsAg/adw, and that although the amino terminus and lysine residues 141 and 160 of both HBsAg/adw and HBsAg/ayw are reactive, their modification does not alter any measurable antigenic activity.     

Antigenic specificity of monoclonal antibodies to human myoglobin

Two monoclonal antibodies directed against different sites of the human myoglobin molecule have been tested for their cross-reactivities against several myoglobins including seven from mammalian species. The relation between their cross-reactivities and their amino acid sequences had led to a possible localization of two antigenic domains in human myoglobin. Each domain includes residues previously considered not to be directly involved in the antigenic structure of myoglobin. Unlike polyclonal serum antibodies, monoclonal hybridoma antibodies directed to a native protein often fail to bind to supposedly antigenic protein fragments. This is explicable in terms of the concept of antigenic domains. Such domains are numerous and overlapping, each comprising a number of contributory amino acid side chains which need not necessarily include continuous sequences of amino acids and which need not exhibit measurable antigenicity in isolation from the rest of the domain.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Summary Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.