A local antigenic determinant distribution in a continuous antigenic region at residues 38-54 of hen egg white lysozyme

The precise location of the antigenic determinants in a continuous antigenic region at residues 38–54 of hen egg white lysozyme (lysozyme) was investigated using the inhibition test of binding of N^α-[¹⁴C]acetyl fragment 38–54 with goat (three individuals) and sheep (four individuals) anti-lysozyme antisera by various synthetic and proteolytic fragments of lysozyme. From these inhibition studies, we found that in this region there were three independent antigenic determinants, consisting of residues 38–45, 40–48, and 44–54, respectively. The existence and the specificity of the antibodies directed to these determinants were further examined with isolating the specific antibodies by affinity chromatography on columns to which the fragment 38–45, 44–48, and 46–54 were bound. The results indicated that these determinants partially overlapped one another in amino acid sequence, but the antibodies directed to them could recognize only each corresponding determinant. These antibodies were also shown to be reactive with native lysozyme as well as a reduced and S-carboxymethylated derivative of lysozyme, and to be found in goat and sheep anti-lysozyme antibodies. The amounts of these antibodies calculated from the binding capacities were in the range from 0 to 48 μg/ml of antisera. These values corresponded to a small fraction of the total precipitable anti-lysozyme antibodies and were as high as 0.8% of the total. The ratios of the amounts of these antibodies differ in individuals or in different species of animals. The binding affinities of N^α-[¹⁴C]acetyl fragment 38–54 with these antibodies were in the range from 1.3 × 10⁷ to 2.6 × 10⁸m^?1. The double-reciprocal plots of the antigen binding with these antibodies drew almost a straight line compared with those of a mixture of several antibody populations, that is, whole antisera.     

Chemical and immunological characterization of proteoglycans of embryonic chick calvaria

We have previously demonstrated in quail embryos grafted on chick yolk sacs the existence of intraembryonic stem cells responsible for definitive hemopoiesis. In order to determine the origin of these cells, we now examine the diffuse hemopoietic processes within the avian embryo's mesoderm. At 4–5 days of incubation in the two species, basophilic cells were found throughout the dorsal mesentery. At 6–8 days these cells became very numerous and built up dense foci at the level of branching of the anterior and posterior cardinal veins. These cells often infiltrated the wall of lymph spaces and channels and were also present in the lumen of blood vessels. Such locations support the interpretation that these basophilic cells represent early stages of hemopoietic differentiation. At 8–10 days, erythropoiesis or granulopoiesis was seen in the foci, which then regressed rapidly. The foci maximal development coincided with the period of colonization of the intraembryonic organ rudiments. In “yolk sac chimeras,” the foci were always constituted by quail cells, indicating their intraembryonic origin. The primordial origin of the intramesodermal cells remains to be determined. A likely source might be the ventral wall of the aorta which appeared to shed cells into the lumen and into the mesentery in the 3-day embryo.     

Chemical characterization of a new surface antigenic polysaccharide from a mutant of Staphylococcus aureus

A mutant of Staphylococcus aureus H was isolated by virtue of its inability to agglutinate with antibodies against teichoic acid of S. aureus. Immunological studies revealed that the mutant, S. aureus T, possessed a new surface antigen in addition to having the antigenic determinant of the wild-type strain, the ribitol teichoic acid. The presence of this additional surface component rendered strain T resistant to staphylococcal typing phages, presumably by masking the phage-receptor sites. The polymer was separated from teichoic acid by chromatography on diethylaminoethyl cellulose and was shown to be composed of two amino sugars, N-acetyl-d-fucosamine and N-acetyl-d-mannosamin uronic acid.     

Chemical and immunological characterization of a novel amphipathic antigen from biotype B Streptococcus sanguis

A new type of amphipathic antigen was extracted from whole cells of Streptococcus sanguis ATCC 10557 (biotype B, serotype II) by the phenol/water method. The extract was treated with nuclease P1, and was applied to a column of Sepharose 6B. Each fraction was checked by passive haemagglutination (PHA) and immunodiffusion tests against anti-10557 serum which was obtained by immunizing rabbits with whole cells of strain ATCC 10557. Strong PHA activity was demonstrated in the first hexose-containing peak (peak 1) eluted near the void volume, while the second hexose-containing peak (peak 2) produced a heavy band against anti-10557 serum in an immunodiffusion test. The third peak (peak 3) which partially overlapped with peak 2 reacted with concanavalin A, but not with the antiserum, in agar gel. Peaks 2 and 3 had no PHA activity. Peak 1 contained only 1% phosphorus, indicating that cells of strain ATCC 10557 possess an amphipathic antigen which differs from the lipoteichoic acids that are common in many Gram-positive bacteria. Peak 1 was a fatty acid-substituted heteropolysaccharide composed of glucose, galactose, mannose, glycerol and fatty acids in a molar ratio of approximately 1.0:1.3:2.7:0.3:1.0. PHA activity was inhibited in the presence of polymerized mannose. Peak 2 was composed of glucose, galactose, rhamnose and N-acetylgalactosamine in a molar ratio of approximately 1.0:1.4:0.8:0.8, which was essentially identical to the serotype II carbohydrate antigen reported previously.     

Molecular cloning and characterization of an antigenic protein with a repeating region from Clonorchis sinensis

In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography. Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one year. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.     

Mapping the antigenic epitope for a monoclonal antibody against lysozyme

A monoclonal antibody (HyHEL-5), prepared to chicken lysozyme c by the method of K?hler and Milstein, identified an antigenic site (epitope) that was shared by the lysozymes of seven different species of galliform birds. The lysozymes of two galliform species, bobwhite quail and chachalaca, shared only partial antigenic identity with the epitope defined by this antibody. Duck lysozyme did not react with the antibody at all. Amino acids that determined the epitope structure were tentatively identified by comparing the amino acid sequences of these lysozymes and assuming the antigenic changes produced by evolutionary substitutions are not due to long-range conformational changes. Arg 68 was identified as a determining amino acid. Arg 68 is hydrogen-bonded to Arg 45, and together these two amino acids form a basic cluster that may be a subsite of the epitope. The antibody inhibited lysis of Micrococcus lysodeikticus by chicken lysozyme. Additionally, Biebrich Scarlet, a dye that binds to the catalytic site, inhibited antibody binding to this lysozyme, which indicates that the epitope extends into the cleft region between Arg 45 and Arg 114. The epitope was hypothesized to involve a region measuring at least 13 x 6 x 15 A including the Arg 68-Arg 45 complex that borders the enzymatic catalytic site. Four other monoclonal antibodies to lysozyme have been partially characterized; each had a distinct pattern of binding specificity for various species of bird lysozymes.     

Enzymatic and immunochemical properties of lysozyme—Restriction of recognition of lysozyme antigenic determinants in pigs

Pigs immunized with lysozyme responded by producing only nonprecipitating antibody throughout the immunization period. Fig antilysozyme antibodies were found to be resistant to papain fragmentation, only 33% of the antibodies were fragmented with papain. From the binding of fluorescein labeled or ¹⁴C-labeled lysozyme to antilysozyme antibodies it was concluded that the antibodies elicited in pigs recognized only two antigenic determinants of lysozyme. These results were confirmed from the binding of Fab fragments to ¹⁴C-lysozyme. Fab fragments prepared from precipitating rabbit antilysozyme antibody bound ¹⁴C-lysozyme at a molar ratio of Fab/lysozyme = 3. Therefore nonprecipitating antibodies are the outcome of recognition of only two antigenic determinants on lysozyme and inability to form a lattice structure when antibody and antigen interact. This work emphasizes the limitations of using antibodies as a biological reagent for delineating the antigenic determinants on proteins.     

Chemical and immunological characterization of developmentally expressed chicken erythroid surface membrane antigens

The expression of two hematopoietic-lymphoid membrane antigens, referred to as chicken fetal antigen (CFA) and chicken adult antigen (CAA) were investigated on primitive and definitive peripheral red blood cells (RBC) from different-aged chickens using chemical and immunological techniques. Differential adsorptions of antisera specific for adult RBC membrane antigens permitted the serological dissection of CAA into eight antigenic determinants. CFA and CAA were assayed by hemagglutination, hemolysis, and immune precipitation of radioiodinated surface antigens of RBC from different-aged chickens. Primitive RBC express CFA, while definitive RBC express three distinct phenotypes: CFA, both CFA and CAA, or CAA, depending on the developmental age of the chicken from which the RBC were obtained. When solubilized membrane extracts of radioiodinated peripheral RBC from chickens at 5 and 18 days embryonic development (E5 and E18, respectively), 13 days posthatch development (H13), and adult chickens were immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the major antigen detected by anti-CFA sera was associated with proteins having apparent molecular weights (M_r) of 50,000 daltons (50 kd). The antigens detected by anti-CAA sera were associated with proteins having apparent M_r of 102, 81, 48, and 43 kd.     

A mycelial mutant ofParacoccidioides brasiliensis defective in dimorphism: Chemical and immunological characterization

A mutant of the dimorphic, human pathogen,Paracoccidioides brasiliensis, blocked in its ability to transform from the mycelial to the yeast form when grown at 37°C, was isolated from a wild-type strain by treatment with nitrosoguanidine. Immunological identity of the mutant with the parental strain was established by means of the species-specific antigen E₂ present in both strains. Cell wall analysis revealed that although a mycelial form was always expressed, differences existed in the chemical structure of walls isolated from cells grown at room temperature and 37°C. The main ones involved the distribution of components (i.e., sugars, amino acids, and amino sugars) in an alkali-insoluble fraction and in the neutral sugar distribution within the same fraction. Although the mutant grew in the mycelial form at 37°C, it was unable to multiply or to transform to the yeast form in experimentally infected animals. This behavior was associated with the absence of α-1,3-glucan in the cell wall of the mutant.     

Radioimmunochemical characterization of hemoglobins Lepore and Kenya: unique antigenic determinants located on hybrid hemoglobins.

Antisera were produced in rabbits to the three known types of Lepore hemoglobins, which contain hybrid delta-beta non-alpha-chains, and to hemoglobin Kenya, which has a hybrid gamma-beta non-alpha-chain. By using a sensitive radioimmunoassay technique, the absorbed antisera were shown to contain an antibody population that was specific for the hybrid hemoglobin and did not cross-react with normal hemoglobins. However, with the absorbed Lepore-specific antisera, the three known types of Lepore hemoglobins were antigenically indistinguishable from each other, suggesting that antibodies are not produced to the primary structural differences which define the three non-alpha-chains of the Lepore hemoglobins. These studies demonstrate that the non-alpha-subunits of hemoglobins Lepore and Kenya possess unique antigenic determinant sites, evidently resulting from an altered polypeptide conformation.     

Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase

The ADP-ribosylation factor (ARF) is a 21-kDa GTP-binding protein cofactor in the cholera toxin-catalyzed ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase. Purified bovine brain ARF was digested with cyanogen bromide, and peptides were purified and sequenced. Approximately 25-30% of the protein was sequenced in this manner. Peptides contained consensus sequences for GTP-binding proteins but were distinct from any of the previously published GTP-binding proteins. Antibodies were raised in rabbits against both protein and synthetic peptide fragments of ARF. Specific ARF immunoreactivity was detected in every eukaryotic tissue or cell examined, including yeast, slime mold, and man. No ARF immunoreactivity was observed when Escherichia coli proteins were tested. Immunoblotting revealed the majority of ARF to be present in the 100,000 x g supernatant. Immunological cross-reactivity with the cytosolic factor indicate that it and ARF are likely to be the same protein. ARF is shown to be myristylated at the amino terminus. The potential role of myristylation in cellular localization is discussed.     

The precise and entire antigenic structure of native lysozyme.

The exact boundary, residue, conformational and directional definitions of the three antigenic sites of native hen's egg-white lysozyme are described. The results clearly reveal that the three antigenic sites account quantitatively for the total antigenic reactivity of the protein. Thus the entire antigenic structure of lysozyme has now been precisely determined and is briefly discussed here, together with the power of the surface-stimulation synthetic concept.     

Binding with lysozyme of antibodies against surface-simulation peptides representing the lysozyme antigenic sites.

Previously it had been shown that native lysozyme has three discontinuous antigenic sites (comprising spatially adjacent residues that may be distant in sequence) that were mimicked by surface-simulation synthetic peptides that had the capacity to bind the bulk (97-99%) of the antibody response against native lysozyme. In the present work these three surface-simulation synthetic peptides were coupled to succinoylated bovine serum albumin, and the conjugates were injected into rabbits. Antibodies against each peptide reacted, as expected, only with that peptide, but it was also found that the antibodies could bind with lysozyme, and the complete specificity of this binding was rigorously established. The advantages of these findings in conformational and immunological investigations are outlined.