Are 'new' antigenic determinants exposed on aggregated bovine serum albumin?

Antigenic specificities of native monomer, heat-denatured monomer and heat-polymerized BSA have been compared serologically. Distinct differences between monomer and polymer preparations were observed with respect to their ability to inhibit each other's binding to antibody and also between their respective rates of dissociation from immune complexes. While it would be possible to interpret the results in terms of the appearance of 'new' antigenic determinants on the polymerized antigen, the results are best explained by an enhanced capacity of polymerized BSA to establish polyvalent binding to antibody of low intrinsic affinity.     

Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin

The aim of this study was to investigate the recognition pattern of bovine serum albumin (BSA), a major dietary protein by serum IgG and IgA antibodies. Anti-BSA IgG and IgA antibodies were measured by ELISA technique in 3 different cohorts: 578 unselected persons, 84 new-onset insulin-dependent diabetes mellitus (IDDM) patients and 103 atopic persons. In order to characterize the recognition pattern of the different BSA domains, recombinant BSA and recombinant fragments covering the 3 BSA domains were produced. BSA digestion was monitored in simulated gastric fluid experiments by means of domain specific monoclonal antibodies. IgG and IgA antibody titres to native BSA were highest in IDDM patients. The three major BSA domains were equally well recognized by IgG antibodies of the three cohorts. Interestingly all three study groups showed a dissociation of their IgG and IgA antibody response to the first BSA domain. The ratio of IgG to IgA antibodies recognizing this domain was 93%/42% in controls, 92%/37% in IDDM patients and 80%/47% in atopic persons. In simulated gastric fluid experiments, the first BSA domain was the first to become undetectable to specific monoclonal antibodies during digestion. In conclusion humoral IgG and IgA antibodies recognize the major BSA domains with different frequencies. The N-terminal domain of BSA, the first to be degraded during simulated gastric digestion is less well recognized by IgA antibodies. This suggests that early digestion is negatively correlated to the IgA antibody response and that the IgA response associated to the gut associated lymphoid tissue (GALT) and the systemic IgG antibody responses are independent.     

Allergenic and antigenic properties of bovine serum albumin

The antigenic sites on bovine serum albumin were studied utilizing peptic and tryptic fragments of the molecule. Rabbits were immunized with small doses of bovine serum albumin and their serum antibody response measured by a bincling assay and the IgE response by antigen-induced histamine release from their basophils. The basophils from two bovine serum albumin allergic individuals were also used for histamine release studies. Serum antibodies bound large fragments of bovine serum albumin; these fragments also induced histamine release from basophils. Although about half of the antibody bincling activity was recovered in the two halves of the albumin molecule only about 10% of the histamine releasing activity was present in the same fragments. The loss of activity of the bovine serum albumin molecule on proteolytic cleavage into two halves could be due to the breakup of the molecule in the middle of the third domain with loss of antigenic sites and/or due to minor conformational changes in the fragments as compared to the intact molecule. Large fragments of bovine serum albumin induced basophil histamine release, thus demonstrating the presence of at least two antigenic determinants on each of these peptides. This data therefore suggests the presence of at least four antigenic IgE-bincling sites on the bovine serum albumin molecule. By basophil desensitization experiments unique IgE-reactive antigenic sites were demonstrated on each half of the molecule; however, some of the sites on the COOH-terminal half cross-reacted with antibodies directed towards the NH₂-terminal part of the molecule. The IgE-response of rabbits to bovine serum albumin was specific; there was no cross-reactivity with rat or mouse albumin. The present finclings indicate a substantial loss in the IgE-reactive determinants of bovine serum albumin by cleavage into large fragments.     

Antigenic determinants of bovine serum albumin.

BACKGROUND: Bovine serum albumin (BSA) is one of the most widely studied proteins; its structure is well known and its antigenic characteristics have been described in several papers. The aim of this research was the identification of the BSA antigenic determinants. METHODS: This study was performed using limited proteolysis and an immunoblotting technique, in which a commercial murine antibody and sera from children sensitized to BSA were used. RESULTS: Findings suggest amino acids (aa) 524-598 as an epitopic area for human species. The most critical sequence seems to be aa 524-542, even if it must be included in a longer fragment to be recognized by antibodies. Murine IgG antibodies also recognize fragments contained in the first half (NH(2)-terminal portion) of BSA. CONCLUSIONS: The results presented in this study indicate that the epitopic sites of an antigenic protein can be different when different species are considered, so that data obtained with antibodies from animal species cannot be directly extrapolated to the behavior of human IgEs.     

Immunochemistry of serum albumin. X. Five major antigenic sites of human serum albumin are extrapolated from bovine albumin and confirmed by synthetic peptides

In a recent report from this laboratory, we have predicted and confirmed by synthesis the locations of five major antigenic sites of bovine serum albumin. In view of the high structural similarity between bovine and human serum albumins, analogous regions of human serum albumin are predicted here to comprise antigenic sites in this protein. Inimunochemlcal studies with antlsera to human albumin and the synthetic antigenic sites of bovine albumin verified this prediction and also identified the major structural locations responsible for the immunochemical cross-reaction between these two albumins.     

Cooperation of antigenic determinants in intact mice and interference by antigenic competition

Cooperation between two immunopotent regions (Phe, G) and Pro-L, of a synthetic polypeptide, multichain (Phe, G)-Pro-L, in a secondary response in intact mice 1s described. (Phe, C) behaves as a haptenic group for which the multichain polypeptides, Pro-L and A-L, act as carriers. When C3H/HeJ mice were primed with (Phe, G)-A-L and challenged with (Phe, G)-Pro-L, a strong carrier effect could be demonstrated, 2.e. the response to (Phe, G) was very poor compared with that obtained upon secondary challenge with (Phe, G)-A-L. Since (Phe, G) is larger than the known size of an antibody combining site, this result argues against a local environment contribution to the carrier effect. Mice received, primary inoculation with mixtures of (Phe, G)-A-L and (T, G)-Pro-L in various molar ratios and then were boosted with (Phe, G)-Pro-L. Cooperation in the secondary response was obtained in some cases, as judged by the enhanced anti-(Phe, G) titers, compared with mice primed with (Phe, G)-A-L alone, after challenge with (Phe, G)-Pro-L. However, the molar ratio of (T, G)-Pro-L/(Phe, C)-A-L in the primary immunization was found to be a critical factor, a priming ratio of 1/5 being optimal. With other priming ratios, no cooperation could be demonstrated in the secondary response. This was shown to be due to antigenic competition between (Phe, G) and Pro–L in the primary response, which reduced the degree of priming or memory for the secondary response to either the haptenic or carrier determinants, Thus, antigenic competition may be an important factor to be considered in the design of experiments to investigate cooperation of antigenic determinants.     

Study of an antigenic site of human serum albumin with monoclonal antibodies

Two monoclonal anti-HSA antibodies, HA1 and HA2, have been shown to be specific for a univalent fragment of 6000 mol. wt, F1, located near the C-terminus of HSA (Doven, Pesce & Lapresle, Immunology Letters 3, 365-370, 1981). Both monoclonal antibodies have been shown to react with the same site, which includes the following components: the last small loop of HSA (558-567) the disulfide bridge 514-559 and the residue 570. This site is as available on HSA and F1, but partially masked on the 'Inhibitor' fragment from which F1 derives. Polyclonal anti-F1 antibodies, purified from rabbit sera or mouse ascites by affinity chromatography, react with the same site as HA1 and HA2. However, polyclonal antibodies are heterogenous, most probably because they consist of anti-F1 specific antibodies and of antibodies specific against other parts of the albumin molecule which cross-react with F1. In addition, monoclonal antibodies can recognize the mutation of a single amino acid residue in the albumin molecule.     

Study of the antigenic structure of human serum albumin with monoclonal antibodies

Analysis of the antigenic structure of human serum albumin was undertaken using monoclonal antibodies. Nineteen antibodies were prepared and their specificities were studied using fragments which encompass the whole sequence of the albumin molecule. These antibodies recognized 13 different epitopes which are different from the one previously identified with two other monoclonal antibodies [Doyen et al., Immun. Lett. 3, 365-370 (1981)]. Among those 13 different epitopes, six were overlapping. Four epitopes were located on the N-terminal half of the albumin molecule. One of these required integrity of methionine 87 and the other three were overlapping and located around methionine 123. Eight epitopes were located on the C-terminal half of the albumin. Two of them were within the sequence, 330-422 and 299-496 respectively; the other six appeared to be topographic determinants which were altered or lost in the albumin fragments. A last epitope could not be located on any region of albumin. Four monoclonal antibodies directed against a given portion of the albumin molecule reacted slightly with another part of albumin, thus confirming the existence of an intramolecular cross-reactivity between the different domains of human albumin.     

Antibody production and antigenic specific suppression to bovine serum albumin in uremic rats

Antibody response and antigen specific suppression to bovine serum albumin (BSA) in uremic rats have been investigated. Primary immune response was found to be significantly reduced independent of the route of administration or adjuvant while secondary response was affected only in animals in which a weak adjuvant such as Al(OH)3 was used. Antigen specific suppression of uremic rats was achieved by intravenous pretreatment with BSA or a C terminal fragment (505-582) of BSA. Secondary response in sham operated controls was suppressed by BSA but not by the fragment. There was about five-fold greater retention of the fragments in the uremic rats than in sham-operated controls at a 6 hr or later after i.v. administration.     

Studies on the induction of antibody synthesis against sulfanilic acid in rabbits. I. Effect of the number of hapten molecules introduced in homologous protein on antibody synthesis against the hapten and the new antigenic determinants

The present experiments were carried out in order to elucidate further the following three questions:

• (1) Is there any difference in “helper activity” between the diazotized rabbit serum albumin (RSA) molecule and the bovine γ-globulin (BGG) molecule in the induction mechanism of antibody synthesis to sulfanilic acid (Sulf) in rabbits?
• (2) Is there an optimal hapten density on a homologous carrier molecule for induction of and maximum antibody synthesis against sulfanilic acid?
• (3) Does the hapten density affect the expression of, and antibody synthesis against the new antigenic determinants introduced through the haptencoupling reaction?

Carrier antibody titers against diazotized RSA and BGG in the rabbits were significantly different when measured between day 7 and day 30, the BGG titer being higher. Thus, the higher anti-Sulf antibody response observed in rabbits challenged with Sulf₁₀ BGG compared to the anti-Sulf response to Sulf₁₁ RSA can presumably be related to a higher immunogenicity and thereby a more effective “helper” property of the BGG molecule. Anti-Sulf antibody titers of similar magnitude were observed when using Sulf₁₁ -, Sulf₂₃ -, and Sulf₄₀ RSA as immunogens, Sulf₄ RSA elicitating a somewhat lower anti-Sulf response. The anti-Sulf antibodies produced against the different SulfRSA conjugates showed increasing proportions of 19 S antibodies with increasing hapten density. Antibody synthesis against new antigenic determinants was observed when using Sulf_4-, and Sulf₁₁ RSA as immunogens, not observed when using Sulf₂₃, and Sulf₄₀ RSA. This finding was in agreement with the in vitro reactivity of the SulfRSA conjugates against sheep anti-RSA: Sulf₄ and Sulf₁₁ RSA reacted with anti-RSA, Sulf₂₃, and Sulf₄₀ RSA did not. These results are discussed on the basis of the cellular cooperation hypothesis, and multivalent binding of high density conjugates onto antibody-forming precursor cells is proposed as a mechanism alternative to cellular cooperation.     

Characterization of conformation independent antigenic determinants in the triple-helical part of calf and rat collagen

About 20 per cent of the antibodies in rabbit antisera to native calf or rat collagen exhibited affinity for denatured rabbit collagen and could be isolated by immunoadsorption. Such antibodies reacted with the unfolded α1-chain as well as with the α2-chain of collagen. Inhibition experiments suggested that the two kinds of polypeptide chains are not completely equivalent in their antigenic determinants. These determinants were not significantly influenced by a treatment of native collagen with pronase, a procedure known to remove short, non-helical sequences at both ends of the molecule. The results suggested that the antigenic determinants are conformation independent. They are, however, mainly located in the middle region of collagen, having a rather complex conformational structure.

Cyanogen bromide cleavage of collagen did not impair the serologic activity of these determinants but with one exception none of the individual cyanogen bromide peptides possessed the full activity of the entire α-chain. However, most of the peptides could be agglutinated by the antibodies when put onto tanned red cells. Inhibition studies of these agglutination reactions clearly demonstrated that virtually all of the peptides carry unique antigenic determinants, which occasionally are shared by a few other peptides. Additional evidence for heterogeneity was obtained by further cleavage of the cyanogen bromide peptides with proteases. The minimum number of antigenic determinants thus estimated in calf collagen was nine. Evidence is provided that their structure in most cases does not correspond to sequences of the type Gly-Pro-X.

     

Identification of antigenic determinants on insulin recognized by monoclonal antibodies

The specificities of nine monoclonal antibodies raised to bovine insulin were investigated. The probable binding sites of the antibodies were determined by correlation of cross-reactivity with heterologous insulins and amino acid differences in the primary structures. Most antibodies recognized topographic determinants composed of both A- and B-chain residues but were capable of binding either one or both free chains independently. Only one antibody was completely conformation-dependent. A number of antibodies showed heteroclitic binding to particular insulin variants. All the antibodies were autoreactive in that they recognized rat insulin which has the same primary sequence as the mouse molecule.     

Relative frequencies of homologous recombination between plasmids introduced into DNA repair-deficient and other mammalian somatic cell lines

Twelve mammalian somatic cell lines, some of them DNA damage-sensitive mutants paired with their respective wild-type parental lines, were assayed for their ability to catalyze extrachromosomal, intermolecular homologous recombination between pSV2neo plasmid recombination substrates. All of the somatic cell lines analyzed are capable of catalyzing homologous recombination; however, there is a wide range of efficiencies with which they do so. Five human cell lines display a fourfold range of recombination frequencies, and six hamster cell lines vary almost 20-fold. Linearizing one of the recombination substrates stimulates recombination in all but one of the cell lines. Two of the three paired mutant cell lines display a threefold reduction in their ability to catalyze homologous recombination when compared to their respective parental cell lines, indicating that the mutations that render them sensitive to DNA damaging agents might also play a role in homologous recombination.     

Characterization of bovine serum albumin epitopes and their role in allergic reactions

Objective:  This review provides updated information on conformational and sequential epitopes identified in bovine serum albumin (BSA) and summarizes available data about the role of structural modifications on BSA antigenicity/allergenicity.
Data sources:  Data on beef allergy and BSA antigenicity are reported, with reference both to the basic literature and to clinical results obtained by our group.
Results and discussion:  BSA is an important allergen involved in milk and beef allergy. The presence of conformational epitopes has been suggested by indirect evidence, while at least one sequential epitope has been experimentally identified. The role of structural modifications on BSA antigenicity is discussed as well as the increased tolerance observed in allergic subjects consuming beef as strained (homogenized) and freeze-dried derivatives.
Conclusion:  Study of the molecular characteristics of a known major allergen allows the identification of technological processes that may be capable of improving the tolerance of allergic subjects to a specific food. Even though any hoped for reduced allergenicity must be verified under medical supervision, the use of new products could obviate the need to avoid important foods such as meat in childhood.     

Characterization of antigenic determinants in histoplasmin that stimulate Histoplasma capsulatum-reactive T cells in vitro.

Although antigen-reactive T lymphocytes play a central role in the host response to Histoplasma capsulatum, little is known of the nature of Histoplasma antigens recognized by these cells in vitro. Employing a murine T-cell line and two clones that are reactive with histoplasmin, we examined whether activation of T cells by histoplasmin required the presence of carbohydrate or protein moieties. The approach taken was to modify carbohydrate or protein molecules in histoplasmin by chemical or enzymatic digestion or by lectin adsorption. In parallel, antigen was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to correlate alterations in functional activity with changes in the electrophoretic appearance of histoplasmin. Treatment of histoplasmin with periodate (0.1 M, 0.05 M, and 0.01 M) or with the endoglycosidases N-glycanase and endoglycosidase H sharply diminished the capacity of histoplasmin to trigger responses by T cells. Reactivity of T cells to histoplasmin that had been adsorbed with lectins binding mannose, glucose, or galactose was reduced by greater than 70%; conversely, the responses by T cells to antigen that had been adsorbed with lectins specific for fucose, N-acetylgalactosamine, or N-acetylglucosamine ranged from 82 to 91% of that to control antigen. Proliferative responses by T cells to histoplasmin that had been digested with chymotrypsin, protease, or trypsin were 2 to 43% of control values. The electrophoretic appearance of histoplasmin was modified by some but not all of the treatments. Partially purified H and M antigens triggered proliferation of T cells. Thus, both carbohydrates and proteins must be present to induce optimal responses by T cells. A portion of the carbohydrates is N linked to proteins, and alpha-D-mannose (or alpha-D-glucose) and beta-D-galactose are the sugar ligands of carbohydrate-containing antigens.     

Modulation of the immunogenicity of antigenic determinants by their flanking residues

Much of the attention on protein antigens and their induction of T-cell responses has focused on the nature of the interactions of their `core' determinants with the major histocompatibility complex (MHC) or the T-cell receptor (TCR). Here, Kamal Moudgil and colleagues instead emphasize the vital role of the amino acid residues that flank the core antigenic determinant on the outcome of the response.     

The identification of antigenic determinants by a coupled inhibition-agar diffusion method

The determination of the chemical nature of immunodeterminant groups of carbohydrate antigens has been achieved by a micro method coupling inhibition and double diffusion in agar. This method has been tested with antigens which react with anti-lactose, anti-galactose, anti-N-acetyl-glucosamine and antirhamnose antibodies. The analysis can be performed with as little as 10 micrograms of inhibitor, 0.2 microgram of antigen and 10 micrograms of antibody. The procedure has also been used for the identification of the determinant groups of 2 antigens with a phosphoglycan structure. The determinants of these antigens have been found to be N-acetyl-beta-glucosamine 1-phosphate and beta-glucose 1-phosphate. The glycosyl 1-phosphate units are novel types of antigenic determinants and antigens with such determinants should be useful for investigating the interactions of antigens with homologous antibodies. The specificity of monoclonal antibodies directed at an O-antigen has been determined by use of the coupled method.     

Characterization of the antigenic determinants of the Leishmania infantum histone H3 recognized by antibodies elicited during canine visceral leishmaniasis

In the present study we show that sera from dogs naturally infected with the protozoan parasite Leishmania infantum contain antibodies that specifically react with the parasite histone H3. Using synthetic peptides covering the complete sequence of the protein we located the linear antigenic determinants within the 40 amino-terminal amino acids of the molecule. In addition to the complete form of the protein (rLiH3), two regions of the Leishmania histone H3 were expressed as recombinant proteins: the rLiH3-Nt fragment containing the 39 amino-terminal amino acids and the rLiH3-Ct fragment containing the 90 carboxyl-terminal residues. Competition experiments using the protein fragment rLiH3-Nt as competitor confirmed that the antigenic determinants of histone H3 are confined to the amino-terminal domain. This domain, which is believed to be exposed on the nucleosome surface, is also the most evolutionarily divergent region of the L. infantum histone H3. Visceral leishmaniasis (VL) sera do not react with mammalian histones, an indication that the anti-histone response elicited during Leishmania infection is triggered by the parasite histone. The results of the prevalence of anti-histone H3 antibodies in canine VL sera together with the sequence-specific characteristics of the amino-terminal region of L. infantum histone H3 indicate that the recombinant protein rLiH3-Nt may be of use for diagnosis of canine VL.     

Cross-reactive and unique grass group I antigenic determinants defined by monoclonal antibodies

As part of a study to probe the immunochemical basis for allergenic cross-reactivity among grass pollens, a series of useful reagents has been prepared. The major grass-pollen allergen, designated group I (GpI), was isolated from five grass pollens (meadow fescue, June grass, sweet vernal grass, redtop grass, and perennial ryegrass). The purified GpI antigens were used to immunize individual groups of BALB/c mice. A total of 123 hybridoma-derived anti-GpI monoclonal antibodies (mAbs) was produced. These mAbs were used to evaluate the antigenic relationship among the GpI antigens by means of two types of ELISA. The experiments revealed a high level of epitope diversity and demonstrated a wide range of antibody specificities. A cross-reactivity ELISA was used to identify and compare antigenic determinants on the GpI molecules, and it was possible to define mAbs with specificities unique for the immunizing allergen and other mAbs that cross-reacted with one or more other members of the test panel of allergens. These murine mAbs reflect the range of specificities present in sera from grass-allergic individuals.