An inhibition enzyme immunoassay for myelin basic protein

An improved Enzyme Immunoassay for Myelin Basic Protein is described. Myelin Basic Protein covalently attached to glass balls, and Myelin Basic Protein in samples compete with each other for binding of a peroxidase conjugated anti Myelin Basic Protein antibody. The peroxidase activity on the balls is then inversely proportional to the amount of Myelin Basic Protein in the sample. A detection limit of 0.6 ng/ml is demonstrated for diluent or spinal fluid. For plasma a dilution step increases this to 1.8 ng/ml. Both the coated balls and the peroxidase conjugate are stable for long periods. The assay requires no expensive equipment. Although the assay appears to be valid for subcellular fractions spinal fluid and plasma, successful detection of Myelin Basic Protection peptides in clinical samples may require careful selection of suitable antisera. The assay would be very suitable for eventual use with an appropriate monoclonal antibody.     

Phosphorylation of myelin basic protein by glycogen phosphorylase kinase

The ability of homogeneous glycogen phosphorylase kinase (Phk) from rabbit skeletal muscle to phosphorylate bovine brain myelin basic protein (MBP) was investigated. Phk could incorporate a maximum of 1.9 mol phosphate/mol MBP. The apparent Km and Vmax for Phk phosphorylation of MBP were 27 microM and 90 nmol/min per mg enzyme, respectively. Properties of MBP phosphorylation by Phk are similar to those of phosphorylase as a substrate. Only serine residues of MBP are phosphorylated by Phk. Phosphorylation sites of MBP by Phk are not identical to those by cAMP-dependent protein kinases.     

Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus

In contrast to canonical proteases, myelin basic protein (MBP)-Sepharose-purified IgG from multiple sclerosis (MS) and systemic lupus erythematosus (SLE) patients efficiently hydrolyze only MBP, but not many other tested proteins. It was shown that anti-MBP SLE IgGs cleave nonspecific tri- and tetrapeptides with an extremely low efficiency and cannot efficiently hydrolyse longer oligopeptides corresponding to antigenic determinants (AGDs) of HIV-1 integrase. To identify all sites of IgG-mediated proteolysis corresponding to two AGDs of MBP, we have used a combination of reverse-phase chromatography (RPhC), MALDI spectrometry, and TLC to analyze the cleavage products of two (17- and 19-mer) encephalytogenic oligopeptides corresponding to these AGDs. Both oligopeptides contained several clustered major and minor sites of cleavage. The active sites of anti-MBP abzymes are localized on their light chains, while the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high specificity of MBP hydrolysis. The affinity of anti-MBP abzymes for intact MBP was ～10(3)-fold higher than for the oligopeptides. The data suggest that both oligopeptides interact mainly with the light chain of different monoclonal abzymes of total pool of IgGs, which possesses lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific.     

Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.     

Intraspecies variability in Vipera ammodytes ammodytes venom related to its toxicity and immunogenic potential

Vipera ammodytes is the most venomous European snake, whose venom has been used as antigen for immunization of antivenom-producing animals. Same as venom of any other snake, it is a complex mixture of proteins, peptides and other compounds which biochemical and pharmacological variability has been demonstrated at interspecies and intraspecies level. In this work we demonstrated intraspecific variability between 8 venom production batches using both the conventional and the new methodology. Moreover, in contrast to the literature on different venoms' variability, for the first time we were able to select those biochemical differences that are related to and give information on the venom's toxicity and immunogenicity. We have shown that methods quantifying ammodytoxin (the most toxic compound identified so far in the Vipera ammodytes ammodytes venom) content of the venom clearly distinguish between high and low immunogenic venoms.