Arthur M. Felix

Defining minimal requirements for antibody production to peptide antigens

The role that individual determinants play in modulating the immune response of an organism to a pathogen is often obscured because of the complexity of the pathogen. In order to gain a better appreciation of the role of individual determinants in the immune response, a pathogen may be dissociated into smaller components, for example peptides representing specific epitopes. These isolated components are often poorly immunogenic and historically have required the use of adjuvants to stimulate antibody production. This report defines the minimal essential requirements for antibody production to a peptide in this system. These are the ability to stimulate both B- and T-helper lymphocytes, anchorage in a phospholipid complex and multivalency within the complex. When these conditions are met, no additional adjuvants are necessary. This procedure has allowed us to identify three distinct T-helper cell epitopes from HIV gp160. In addition, this information has been used to produce a simple, totally synthetic and highly immunogenic preparation for the production of antibodies to peptides.

Type-specific and cross-reactive epitopes in human papillomavirus type 16 capsid proteins

Genital human papillomavirus (HPV) 16 infection is frequently associated with cancer of the uterine cervix, as well as with precancerous lesions. In order to generate serologic reagents which might be useful in the diagnosis of HPV 16 infection, rabbit polyclonal and mouse monoclonal antisera were raised to carboxy terminal peptides from the HPV 16 L1 and L2 open reading frames (ORFs). Anti-L1 and -L2 peptide sera recognized HPV 16 L1 and L2 fusion proteins in Western blots and by immunoprecipitation. In Western blot analysis of L1 proteins from different HPV types, antisera to the L1 peptide reacted only with HPV 16, thus identifying an HPV 16 type-specific linear epitope. Anti-L2 peptide sera reacted with L2 fusion proteins from HPVs 6 and 16, but not from BPV, thus identifying a partially cross-reactive epitope in the HPV 16 L2. Computer analysis of carboxy terminal amino acid sequences of the L1 and L2 ORFs of multiple HPV types supported the Western blot findings. Despite the HPV 16 type specificity found in Western blots, anti-L1 peptide sera identified nuclear antigen by immunocytochemistry in cervical biopsies infected with HPV 16, as well as other genital HPV types. Anti-L2 peptide sera failed to recognize antigen in infected tissue.

[33] Analysis of different forms of recombinant human interferons by high-performance liquid chromatography

Publisher Summary This chapter discusses the analysis of different forms of recombinant human interferons by high-performance liquid chromatography (HPLC). The cloning and expression of the recombinant human leukocyte interferon A (IFN-αA) gene in E. coli and subsequent purification by the use of immobilized monoclonal antibodies has led to the scaled-up production of IFN-αA for human clinical trials. Analysis of the resultant IFN-αA by nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of higher oligomers (dimers, trimers, and tetramers) and at least two monomeric forms. The two major monomeric forms have been termed “slow” and “fast” migrating monomers (SMM and FMM, respectively) to designate their relative mobilities on SDS-PAGE. Although the components of IFN-αA can be separated by SDS-PAGE, HPLC is an alternative method, which may be advantageous for the routine analysis of multiple samples. The HPLC system comprises of two constametric pumps (Models I and IIG), Analytical HPLC is a powerful tool for the evaluation of human leukocyte interferons (IFN-αA, IFN-αD), hybrids (IFN-αA/D), immune interferon (IFN-γ) as well as related synthetic peptide fragments. The three forms of human leukocyte interferon can be resolved, and chromatograms of admixtures can be readily carried out to make positive identification of known impurities.

Enzymatic Semisynthesis of Growth Hormone-Releasing Factor and Potent Analogs

The potential value of enzymes in peptide synthesis is derived from the highly selective nature of these catalysts. Proteolytic enzymes can catalyze regioselective acylations, thereby eliminating the need for side-chain-protecting groups in favorable cases. The rigid stereoselectivity of the proteases ensures optically pure products, even from racemic starting materials. These properties make biocatalysis especially attractive for convergent synthetic strategies employing segment condensations in which racemization is often a concern with conventional stoichiometric coupling reagents. In addition, enzymatic segment condensations can be performed for segments without the side-chain-protecting groups that often create solubility problems and require additional stages of deprotection.

Derivation Of Solution Conformers Of Peptide Hormones Via Constrained Molecular Dynamics Based On 2-D NMR Data

An optimization procedure based on molecular dynamics and energy minimization constrained by distances from NOE measurements has been developed to derive and analyze peptide conformations. This procedure readily folds a peptide from its fully-extended conformation to an ensemble of conformers satisfying the distance constraints and, in the process, makes self-consistent chiral assignments of pro-chiral protons with split resonances. Statistical analysis of the ensemble yields families with similar overall backbone conformations and reveals which segments of the peptide chain have well-defined single conformations. CD spectroscopy gives percentages of secondary structure in accord with the well-defined segments from the optimization. The structure of the 73-residue blood complement protein porcine CSa(des-Arg) was determined by a combination of model building. NMR data and the molecular dynamics optimization procedure. The protein structure is a bundle of four a-helices; the C-terminal eight residues are completely disordered in solution. The four-helix bundle forms readily in the molecular dynamics optimization starting from a fully-extended chain without any constraints for the disulfide linkages.