Immunochemical study of β-glucuronidase inhibitor from porcine sublingual gland Relationship between the antigenic determinant and the active site of the inhibitor

Following removal of sialic acid by neuraminidase treatment the activity of the β-glucuronidase inhibitor was remarkably decreased, but the antigenic determinant was not affected.A partial common antigen to the inhibitor was isolated from porcine small intestine, by successive fractionation of trypsin extraction of the latter on Sephadex G-150, DEAE-cellulose and Sepharose 4B column chromatography. The immunologic and characteristic properties of the common antigen were compared with those of the inhibitor, and it was concluded that the active site of the β-glucuronidase inhibitor is not identical with its antigenic determinant.     

Detection of common flagella antigen in Bacillus cereus by monoclonal antibody

Bacillus cereus has been classified into 23 types by immunochemical analyses of flagella antigen, but a common antigenic determinant of flagella had not been determined. When the immunochemical method of classification had changed from "agglutination method" to "enzyme-linked immunosorbent assay (ELISA)," the cross-reactivity between each flagella type was increased. These results indicated that the application of ELISA method would enable detection of the common antigenic determinant of B. cereus. Therefore, we attempted to make monoclonal antibody against common flagella antigen that could be detected by ELISA. Monoclonal antibody provided was specific for B. cereus (H-1 to H-23) and did not show the cross-reactivity with Escherichia coli NIH and Bacillus subtilis.     

Immunological studies on the beef heart natural ATPase inhibitor: localization of an antigenic determinant in the inhibitor molecule

Antibodies were raised against the beef heart mitochondrial ATPase inhibitor. This antiserum prevented the ability of the ATPase inhibitor to inhibit the F1 ATPase activity. Peptide fragments obtained by enzymatic cleavage of the inhibitor protein were tested by immunoblotting or ELISA for their response to the anti-inhibitor antiserum. An antigenic determinant was located in the sequence spanning His 48 to Lys 58 of the inhibitor molecule.     

A mouse plasma cell surface antigenic determinant (PLKB) recognized by xenoantiserum. Its relationship to the PC.1 antigenic determinant.

The specificities of the xenoantisera made against mouse myeloma cells have been compared to those recognized by alloantiserum by studying patterns of cytotoxicity on both normal and malignant plasma cells. Goat antiserum obtained by immunization with Balb/c mouse myeloma ADJ-PC-22A cells and purified by in vivo absorption could detect cell surface antigenic determinants present on plasma cells and on cells of liver, kidney, and brain (PLKB antigen), as we had previously reported for a similarly prepared rabbit antiserum. In spite of an apparent similarity between the tissue representation of the PLKB determinant and that of PC.1 antigenic determinants which were detected by DBA/2 anti-ADJ-PC-22A cell alloantiserum, the PLKB antigenic determinant is not identical with the PC.1 antigenic determinant, since the former is found on the tissues of PC.1-negative as well as PC.1-positive strains of mice. However, it was deduced that the PLKB antigenic determinant and the PC.1 antigenic determinant reside in close proximity on the cell surface or maybe even on the same molecule, since Fab fragments of antiserum against either PLKB or PC.1 blocked the cytotoxicity against both antigens. On the other hand, these Fab fragments did not inhibit the cytotoxicity of anti-H-2 antiserum, indicating that neither PLKB nor PC.1 antigenic determinants are in close proximity to H-2 antigens. Association of PLKB and PC.1 determinants was further supported by the finding that the loss of the PLKB determinant in a variant of myeloma MOPC-70A corresponds to the loss of PC.1 determinant on the same cells.     

Monoclonal antibodies against an alpha-amylase inhibitor from wheat kernel

An alpha-amylase inhibitor (called the 0.53-inhibitor, Maeda, K., Takamori, Y. and Oka, O. (1982) Agric. Biol. Chem. 41, 2873-2875) and the carboxymethylated inhibitor were used to immunize mice (strain BALB/c) according to a procedure described earlier (McMaster, W.R. and Williams, A.F., (1979) Eur. J. Immunol. 9, 426-433). After fusion of spleen cells with NS-1 myeloma cells, three stable clones producing antibodies against the inhibitor were obtained. The binding characteristics of the monoclonal antibodies, AWAI-1, AWAI-2 and AWAI-3, to the inhibitor were analyzed by radioimmunoassay. Two of these monoclonal antibodies to the alpha-amylase inhibitor did not show any binding affinity towards carboxymethylated inhibitor, suggesting that the main antigenic determinant on the native inhibitor is tertiary-structure dependent. The monoclonal antibodies obtained cross-reacted with three other alpha-amylase inhibitors (the 0.19-, the 0.36- and the 0.38-inhibitor) in wheat and these were separated together with the 0.53-inhibitor from the rest of inhibitors by immunoaffinity chromatography. One stable clone producing antibody against the carboxymethylated inhibitor was also established, AWAI-4. The antigenic determinant to this antibody was found to be included in the region of Met(5)-Lys(25) on the carboxymethylated inhibitor.     

Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse. An immunohistochemical study

Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuraminidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.     

Demonstration of multiple antigenic sites of the SV40 transplantation rejection antigen by using cytotoxic T lymphocyte clones

Multiple antigenic sites on the simian virus 40 (SV40) tumor-specific transplantation antigen (TSTA) were detected by the use of cytotoxic T lymphocyte (CTL) clones isolated from continuous cultures of SV40-specific CTL (H-2b). Two independently derived clones, K11 and K19, specific for the SV40 TSTA in association with H-2Db, each recognized a different antigenic determinant of the SV40 TSTA. This conclusion was based on the observation that a human papovavirus BK virus (BKV) transformed cell line, which possesses a T antigen serologically cross-reactive with that of SV40, was lysed by a heterogeneous population of SV40-immune lymphocytes and by clone K19 but not by K11. Therefore, these CTL clones must recognize two different antigenic determinants of the SV40 TSTA:K19 recognizes a cross-reactive determinant of the SV40 and BKV TSTA, whereas K11 is reactive against an SV40-specific determinant.     

Compound d+y+ particles of Australia antigen.

Australia antigen (HB8 Ag) particles vary in their antigenic structure. They are generally found to carry either determinant d or determinant y, but not both. This report describes seven sera which contain Australia antigen carrying both d and y on the same particle.     

Characterization of the antigenic determinant on Fusarium oxysporum recognized by a genus-specific monoclonal antibody

The antigenic determinant of a monoclonal antibody (MAb) (API9-2) having specific reactivity with the fungi grouped into the genus Fusarium was analyzed. The culture supernatant of the fungi showed antigenicity against MAb API9-2, proving that the antigen exists as an exoantigen. The heat-resistant, proteinase K-resistant and periodate oxidation-labile features of the antigenic determinant indicated its carbohydrate nature. Also, lectin affinity tests and thin-layer chromatography analysis suggested that the monosaccharide making up the antigenic determinant was mainly mannose. Considering previous reports that the antigen exists on the surface of mycelia (by immunofluorescence assay) and is a - 55 kDa molecule (by Western blotting analysis), it was concluded that the antigenic determinant of MAb API9-2 on F. oxysporum is a mannan component existing on the surface of mycelia.     

Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.     

Antigenic sites on cytochrome c2 from Rhodospirillum rubrum.

The antigenic determinants for three monoclonal antibodies against cytochrome c2 from Rhodospirillum rubrum were partially characterized by differential chemical modification of free and antibody-bound cytochrome c2 and by cross-reactivity analysis with different antigens. Circular dichroism spectroscopy was used to probe the effect of antibody binding on the conformation of cytochrome c2. The binding of two antibodies was strongly dependent on the native folding of the antigen. The first antibody bound to a determinant around the exposed heme edge on the 'front side' of the molecule which is not antigenic in mitochondrial cytochrome c2. Binding of this antibody to cytochrome c increased the induced CD of the ferric heme in a manner similar to that observed previously when mitochondrial cytochrome-c oxidase bound to the front side of cytochrome c. This observation points to a subtle conformational adaptation of the antigen induced by the antibody. The determinant for the second antibody, which also affected the heme CD spectrum of the antigen, was on a polypeptide loop where cytochrome c2 differs from mitochondrial cytochrome c by an eight-residue insertion. The third antibody, which did not induce a change in CD, bound to a sequential determinant near the amino end of cytochrome c2. Only this antibody cross-reacted with isolated cytochrome-c-derived peptides and with apo-cytochrome c2. A preliminary analysis of the polyclonal immune response of five rats against cytochrome c2 indicates that, unlike in eukaryotic cytochrome c, antigenic determinants are distributed over the whole polypeptide chain of the prokaryotic immunogen.     

Antigens of Pichinde virus I. Relationship of soluble antigens derived from infected BHK-21 cells to the structural components of the virion.

Antigens detected by the complement-fixation (CF) test were prepared from BHK-21 cells infected with Pichinde virus.The preparations contained two antigens demonstrable by immunodiffusion. The antigen present in abundance was heat stable, Pronase resistant, and had a molecular weight of 20,000 to 30,000 as estimated by gel filtration. Polyacrylamide gel electrophoresis of purified antigen demonstrated two low-molecular-weight polypeptides. An identical antigenic determinant was found by disrupting purified virus with Nonidet P-40; however, none of the viral polypeptides co-migrated with the polypeptides derived from purified CF antigen. Pronase digestion of disrupted virus did not alter antigenicity but degraded the viral peptides to sizes similar to those associated with the major CF antigen. These observations suggest that the major CF antigen of Pichnide virus is a cleavage product of the structural proteins of the virus.     

Altered immunochemical reactivity of Saccharomyces cerevisiae a-cells after alpha-factor-induced morphogenesis.

Antibodies were raised against Saccharomyces cerevisiae a-cells that had been exposed to the sex pheromone, alpha-factor. After adsorption of the antiserum with diploid cells, antibodies remained that reacted specifically with the mannan from haploid cells. The characteristic determinant was observed in mannan from pheromone-treated a-cells, in mannan from untreated alpha-cells, and at a much lower concentration, in mannan from control a-cells. The antigens from these three mannans appeared to be identical. The determinant was destroyed by mild-acid hydrolysis or periodate oxidation, but not by proteolysis or digestion with exo-alpha-mannanase. Mutants with altered mannan were unable to express the antigen. Complete acid hydrolysates mannan from alpha-factor-treated a-cells contained mannose, glucose, and N-acetylglucosamine. Partial acid hydrolysis, under conditions that destroyed the antigenic determinant, released only mannose and mannobiose. The mannose fraction was labeled to high specific activity during response of a-cells to alpha-factor if radioactive glucose was the carbon source. Neither alpha- not beta-D-mannopyranosyl phosphate was a hapten. The results are consistent with the presence of a haploid-specific antigen containing an acid-labile mannose determinant and show that the amount of this antigen in a-cell mannan is increased in response to alpha-factor.     

Cryptodeterminant of a sperm maturation antigen on the mouse flagellar surface.

The determinant of a mouse sperm maturation antigen was examined morphologically and biochemically with monoclonal antibody T21 as a probe. The plasma membrane components of cauda epididymal spermatozoa were extracted with nonionic detergent Nonidet P-40 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and by immunoblotting. Wheat germ agglutinin-lectin staining and immunoblotting indicated that the antigen recognized by T21 is a sialoglycoprotein of about 54,000 daltons (54 kDa). The antigenic determinant was more distinctly exposed after treatment with neuraminidase, as evaluated by immunohistochemistry, immunocytochemistry, and immunoblotting. The cryptic nature of the determinant was further confirmed by immunostaining nitrocellulose strips, subsequently digesting the strips with neuraminidase, and then reimmunostaining them. Results obtained by periodate oxidation treatment suggested that the epitope is a carbohydrate. Immunoperoxidase electron microscopy confirmed that the antigen is distributed on the flagellar plasma membrane of the sperm. This was demonstrated clearly when sperm were desialylated with neuraminidase. These results indicate that the 54 kDa sialoglycoprotein sperm maturation antigen has a cryptodeterminant which can be masked by a sialic acid residue, that is recognized by monoclonal antibody T21.     

Mechanism of antigenic competition. II. Induction by specific T cell products

Since previous work had indicated that the mechanism of antigenic competition induced by the injection of red cells was due to a reversible block of cooperation at the surface of the macrophage, the possibility that the inhibitor was complexes of IgT and antigen resulting from the previous response (to red cells) was investigated. Macrophages were incubated with supernatants from activated T cells (ATC), containing, or deprived of, IgT by incubation with anti-mouse Ig-coated sepharose beads. Macrophages incubated with ATC supernatants were immunogenic for the corresponding antigen, but were not capable of binding a second ATC supernatant, of different antigenic specificity, in an immunogenic way. In contrast, if IgT was previously removed, the macrophages were not immunogenic and were not blocked. These results support the concept that this form of antigenic competition is due to blockage of macrophage receptor sites for IgT.     

Monoclonal antibody localization of sperm surface antigen secreted by the epididymis of the baboon (Papio cynocephalus)

A monoclonal antibody (BSA6) was generated against an antigenic determinant secreted by the epididymis of the baboon and present on the acrosomal surface of the spermatozoa. This determinant was first secreted by the principal cells of the proximal corpus region, as determined by fluorescent microscopy performed on Bouin-fixed epididymal tissue sections. The secretory product subsequently bound on the lateral acrosomal surfaces in the distal corpus region, but became uniformly distributed over the acrosomal region in the cauda epididymidis. The antigenic determinant had a molecular weight of 82,000 (western blot technique). The testis, caput and other somatic tissues were devoid of the antigen, indicating the restriction of the antigen to spermatozoa and epithelial cells of the corpus epididymidis. Examination of similar tissue from immature baboons indicated that the secretion of this antigen was age-dependent, secretion beginning at about 4 years of age.     

Regions of the Streptococcus sobrinus spaA gene encoding major determinants of antigen I.

Surface protein antigen A (SpaA), also called antigen B, antigen I/II, or antigen P1, is an abundant cell envelope protein that is the major antigenic determinant of Streptococcus sobrinus and other members of the Streptococcus mutans group of cariogenic bacteria. This laboratory has previously reported the cloning and expression in Escherichia coli of a BamHI restriction fragment of S. sobrinus DNA containing most of the spaA gene (pYA726) and encoding antigen I. Regions of spaA encoding immunodeterminants of antigen I were analyzed by either deletion mapping or expressing selected restriction fragments from the trc promoter. SpaA proteins produced by mutants harboring nested deletions, constructed by BAL 31 exonuclease treatment at a unique SstI site located towards the 3' end of the gene, were examined by Western immunoblot with rabbit serum against SpaA from S. sobrinus. Only SpaA polypeptides larger than 56 kilodaltons reacted with anti-SpaA serum. Various restriction fragments of the region of spaA encoding the antigenic determinants were cloned into an expression vector. The immunoreactive properties of the polypeptides encoded by those fragments indicated that expression of the immunodominant determinant required topographically assembled residues specified by noncontiguous regions located within 0.48-kilobase PvuII-to-SstI and 1.2-kilobase SstI-to-HindIII fragments which were adjacent on the spaA map.     

Turtle-dove ovomucoid, a glycoprotein proteinase inhibitor with P1-blood-group antigen activity.

Ovomucoid from the egg white of turtle-dove (Streptopelia risoria) was purified and shown to be a glycoprotein of mol. wt. 29 400, with valine as N-terminal residue. It is an inhibitor of both trypsin and chymotrypsin, but has a lower affinity for trypsin than has hen ovomucoid. Turtle-dove ovomucoid contains antigenic activity cross-reacting with the blood-group-P1 antigen of human erythrocytes. Hen ovomucoid has no detectable blood group-P1 activity. The carbohydrate composition of turtle-dove ovomucoid differs from hen ovomucoid in having substantially higher galactose content. The possible relationship between carbohydrate composition and antigenic activity is discussed.     

Fluorescence study on the conformation of a cyclic enkephalin analog in aqueous solution

Certain patients with ovarian germ cell tumors develop a specific antibody reacting with glycoprotein-bound large carbohydrates of murine teratocarcinoma cells. The antigenic determinant was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases abolished the antigenic activity of the large glycan isolated from F9 and OTT6050 cells. Several evidences excluded the possibility that the antigen is blood group B or P1 antigen. These results indicate tumor-associated expression of an unusual alpha-galactosyl residue in human ovarian germ cell tumors.     

Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins

Four monoclonal antibodies that are directed against antigens present in sperm and absent from other worm tissues were characterized. Antibody TR20 is directed against the major sperm proteins, a family of small, abundant, cytoplasmic proteins that have been previously described (Klass, M. R., and D. Hirsh, 1981, Dev. Biol., 84:299-312; Burke, D. J., and S. Ward, 1983, J. Mol. Biol., 171:1-29). Three other antibodies, SP56, SP150, and TR11, are all directed against the same set of minor sperm polypeptides that range in size from 29 to 215 kD. More than eight different sperm polypeptides are antigenic by both immunotransfer and immunoprecipitation assays. The three antibodies are different immunoglobulin subclasses, yet they compete with each other for antigen binding so they are directed against the same antigenic determinant on the multiple sperm proteins. This antigenic determinant is sensitive to any of six different proteases, is insensitive to periodate oxidation or N-glycanase digestion, and is detectable on a polypeptide synthesized in vitro. Therefore, the antigenic determinant resides in the polypeptide chain. However, peptide fragments of the proteins are not antigenic, thus the determinant is likely to be dependent on polypeptide conformation. The antigenic determinant shared by these proteins could represent a common structural feature of importance to the localization or cellular specificity of these proteins.