Sydney J. Leach

A novel method for delineating antigenic determinants: Peptide synthesis and radioimmunoassay using the same solid support

Abstract A new method for the rapid assessment of the antigenicity of small peptides has been developed using a solid support which permits both the synthesis of the peptide by conventional solid-phase techniques and the assessment of its antigenicity by a radioimmunoassay procedure. The method has been tested by the stepwise synthesis of a peptide corresponding to a known antigenic determinant of myoglobin followed by radioimmunoadsorption of specific anti-myoglobin antibodies at each stage of the synthesis.

The immunogenicity and antigenicity of proteins

Abstract The exploration of antigenic regions on the surface of proteins and viruses has become easier with the advent of monoclonal antibodies and rapid methods peptide synthesis. For small proteins of known sequence and conformation - myoglobin, cytochrome c, insulin and lysozyme - the antigenic sites may be described as surface domains made up from amino acid side chains which may be distant in sequence but close in space. Such domains are probably overlapping and cover most of the protein surface.

Elimination of nonspecific adsorption of serum proteins by Sepharose-bound antigens

Abstract Nonspecific adsorption of serum proteins occurs with immunoadsorption of antibodies on Sepharose-myoglobin and Sepharose-staphylococcal nuclease immunoadsorbents. This adsorption results from nonspecific hydrophobic and ionic interactions between these serum proteins and the immunoadsorbents. Various preelution washing procedures were examined, and only borate-saline buffer (pH 8,5) containing a nonionic detergent, Tween 20 (0,1%), and a high salt concentration (1 m NaCl) eliminated or significantly reduced nonspecific adsorption without appreciably diminishing the recovery of specifically adsorbed antibodies.

On topographic antigenic determinants in myoglobins

Abstract A study of the antigenic cross-reactivities of beef, sheep and pig myoglobins is reported, in which attention is focused on a single determinant. The amino acid sequence of this determinant (residues 15–22) is identical in beef and sheep myoglobins and only six other residues differ in the rest of their 153 residues. An anti-beef myoglobin antibody fraction which binds specifically to beef myoglobin peptide (1–55) is shown to contain some antibodies which bind equally well to beef and sheep myoglobins and other antibodies which bind more strongly to the beef protein. The evidence from this and several other sources is discussed in the light of current hypotheses on the topographic nature of antigenic determinants.

Two large immunogenic and antigenic myoglobin peptides and the effects of cyclisation

Peptides corresponding to sequences (72-88) and (26-54) of beef myoglobin have been synthesised in their open-chain and cyclised forms (using a disulphide bridge) and tested for their antigenicity and immunogenicity. Antibodies raised to beef myoglobin bound to both peptides but more strongly to the 29-residue than to the 17-residue peptide. Cyclisation increased the antigenicity of the larger peptide. In this form the peptide competed much more strongly than in the uncyclised form for specific antibodies to beef myoglobin. The peptides are immunogenic in mice without being coupled to a protein carrier and produce antibodies which bind to beef myoglobin. Peptide (26-54) is the more immunogenic in producing a larger antibody titre to the parent myoglobin and cyclisation again enhances this property. The findings lend weight to the view that longer peptide sequences might be expected to favour the folded state, therefore binding more strongly to antibodies raised to the native protein and eliciting a population of antibodies which contain a larger proportion specific for that conformation. Cyclisation enhances antigenicity and immunogenicity presumably by decreasing the number of degrees of conformational freedom of a peptide without excluding native-like conformations.

Cross-reactivity between mammalian myoglobins: Linear vs spatial antigenic determinants

Abstract Cross-reactivities between myoglobins from sperm whale, horse, pig and beef have been measured by radioimmunoassay procedures. Taking into account the high degree of homology of the amino acid sequences in the regions believed to be antigenic in the four proteins, the effect of amino acid substitutions outside these regions is unexpectedly large. The spatial character of antigenic domains in proteins is discussed in the light of these findings.

The detection of five antigenically reactive regions in the soybean leghemoglobin a molecule

Abstract By comparison with the amino acid sequence of the sperm whale myoglobin molecule five antigenicaliy reactive regions on the surface of the soybean leghemoglobin a molecule were predicted. The reactivity of these five regions was substantiated using the approach of Smith et al . (1977) in which peptides corresponding to these regions of the molecule were synthesized by a solid-phase procedure on a macroporous resin. At each step of the synthesis the peptides were tested for antigenicity using 125 I-labelled anti-(leghemoglobin a) antibodies whilst still bound to the solid support.

The antigenicity of myoglobin-related peptides synthesised on polyacrylamide and polystyrene resin supports.

Polyacrylamide resins [Atherton et al., Bioorg. Chem. 8, 351-370 (1979)] have been found suitable for solid-phase radioimmunoassay of peptides synthesised on the same supports; they are sufficiently stable during side-chain deprotection and swell sufficiently in aq. media to admit antibody molecules to the sites of peptide attachment. A re-examination of five synthetic peptide sequences corresponding to (15-21), (56-62), (94-99), (113-119) and (145-151) of beef myoglobin analogous to those delineated by Atassi [Immunochemistry 12, 423-438 (1975)] for sperm whale myoglobin shows that they all bind anti-beef myoglobin antibodies raised in rabbits, with binding capacities in the order V = III greater than IV greater than I = II. The resin-bound peptide (72-88) binds such antibodies even more extensively, as do certain sequential variants of peptide V. Other peptides, bound to polyacrylamide or polystyrene resins but unrelated to any of the five sequences and varying in size and amino acid composition and sequence were also tested with various antisera. It was concluded that the antibody binding properties of the 30 or so small peptides (two-seven residues) are dominated by their cationic and/or hydrophobic properties. In small peptides, therefore, antibody binding can be safely interpreted only in terms of general structural properties but not in terms of biological specificity. The latter property becomes assessable only with peptides representing larger areas of antigenic protein surfaces.

THE AMINO ACID SEQUENCE OF SOYBEAN LEGHAEMOGLOBIN c2

1. Introduction The leghaemoglobin found in the root nodules of the soybean plant (G&&e max cultivar Lincoln) after rhizobial infection was originally fractionated into two major and two minor components [ 1,2]. Recently, the second of the major components, leghaemoglobin c, has been found to consist of two distinct molecular species, leghaemoglobin cl and c2, separable by elution from a DEAE cellulose column using a continuous acetate gradient at pH 5.2 [3]. Since the amino acid sequence of the first major component, leghaemoglobin a, is known [4] and it has been well characterized both structurally, and functionally [S-8] we felt it would be of value to elucidate the primary structure of a second major component, leghaemoglobin c2, as a step towards establishing a function (if any) for the multiplicity of leghaemoglobin species found in the root nodules of most legumes. 2. Materials and methods Soybean plants were grown as previously described [9] and isolated by gradient elution (10 to 50 mM acetate) at pH 5.2 from a DEAE cellulose column [3]. The leghaemoglobin c2 fraction was pooled and rechro- matographed on a shallow gradient (25-35 mM acetate) to ensure removal of the cl component. The protein (65 mg) was dehaemed by the HCl-acetone method [lo] and succinylated in an auto-pH titrator at pH 9.2 by the addition of eight lots of

Original antigenic sin: experiments with a defined antigen.

Abstract The phenomenon referred to as ‘Original Antigenic Sin’ is the production of antibodies to a specific antigen elicited by a boosting injection of a related antigen. We have investigated this phenomenon using myoglobins of known amino acid sequence and structure. Our results indicated that in order to elicit an ‘Original Antigenic Sin’ response the boosting myoglobin should differ by less than 33–42% in overall sequence from the priming myoglobin. The antibodies in the antisera from rabbits primed with beef myoglobin and boosted with a different myoglobin from a different species are predominantly and perhaps exclusively directed towards antigenic regions common to the priming and boosting myoglobins but have a higher affinity for the priming myoglobin.