Thomas P. Hopp

A computer program for predicting protein antigenic determinants.

A computerized method for predicting the locations of protein antigenic determinants is presented, which requires only the amino acid sequence of a protein, and no other information. This procedure has been used to predict the major antigenic determinant of the hepatitis B surface antigen, as well as antigenic sites on a series of test proteins of known antigenic structure [Hopp & Woods (1981) Proc. natn. Acad. Sci. U.S.A. 78, 3824-3828.] The method is suitable for use in smaller personal computers, and is written in the BASIC language, in order to make it available to investigators with limited computer experience and/or resources. A means of locating multiple antigenic sites on a homologous series of proteins is demonstrated using the influenza hemagglutinin as an example.

Metal-binding properties of a calcium-dependent monoclonal antibody

The calcium-dependent mAb, M1 (also called anti-Flag or 4E11) was studied using a newly developed metal-sensitive enzyme-linked immunosorbent assay (ELISA). This antibody, specific for a calcium complex of the peptide antigen, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, has found widespread use as a mild purification reagent for Flag-epitope tagged recombinant proteins. Although M1 affinity columns release monovalent Flagged proteins in the absence of calcium, the antibody retains substantial affinity for the Flag sequence even in metal-free conditions, so that it has been impossible to use it to develop a metal-sensitive ELISA assay. This is due to the ability of the antibody to remain bound to polyvalent surface-coated antigen, for instance, when Flagged proteins are bound to ELISA plates or blotting filters. The resultant antigen polyvalence raises the avidity of the Flag antibody to a point where the reaction is essentially calcium-independent. However, when the antibody itself was made monovalent, by proteolytic cleavage to the Fab, this situation was reversed and the ELISA reaction became calcium-dependent. This new metal-dependent ELISA assay was used to explore the metal requirements of the antibody in detail. Among divalent metals, binding tapered off with increasing radius above that of calcium, or with decreasing radius below that of calcium. Several smaller metals, such as nickel, acted as inhibitors of the binding reaction. Substantial binding was demonstrated for heavy metals such as cadmium, lanthanum and samarium. Because it is of interest to use this antibody for the co-crystallization of recombinant Flag-fusion proteins, the ability to bind heavy metals was a significant finding.

Immunochemical studies on α-lactalbumin

The antigenic structural features of alpha-lactalbumin have been investigated using a radioimmunoassay, peptide inhibition of the quantitative precipitin reaction, and by immunodiffusion analysis after chemical modification of the molecule. Antigenic activity (in rabbits) was localized to several peptic fragments and the single arginine residue of bovine alpha-lactalbumin. Antigenic activity was also found to be associated with the single methionine residue. A peptic fragment containing a disulfide loop was found to possess antigenic activity in both bovine and goat alpha-lactalbumin. Radioimmunoassay cross-reactivity between the alpha-lactalbumins is correlated with amino acid sequence similarities; bovine alpha-lactalbumin antiserum cross-reacts with goat alpha-lactalbumin more extensively than with human alpha-lactalbumin, while the more distantly homologous protein, chicken lysozyme, does not cross-react at all. Nevertheless our data indicate that the alpha-lactalbumins and lysozyme share a similar distribution of antigenic determinants on their surfaces.

Identification of aqueous phase amino acid phenylthiohydantoins on polyamide sheets

The aqueous phase phenylthiohydantion (PTH) derivatives of arginine, histidine, and S-pyridylethylcysteine are successfully separated by thin-layer chromatography on polyamide sheets using 10% aqueous pyridine as solvent. Trials on fractions from sequenator runs show that this solvent also separates these PTHs from contaminants which are usually present in the aqueous phase. Development with a second-dimension solvent containing a fluorescent indicator provides a method for identifying these PTHs in subnanomole quantities.

Routine Analysis of Low-Picomole-Level Phenylthiohydantoins by HPLC Using a Diisopropylethylamine-Acetate/THF Buffer and Acetonitrile Gradient

The commercial availability of the gas-phase protein sequencer (1, 2) has greatly enhanced the ability of investigators to obtain information from subnanomolar amounts of polypeptide. To take full advantage of this new technology, however, it has been necessary to develop sensitive and efficient separation methods for phenylthiohydantoin (PTH) amino acids. Although reversed-phase high performance liquid chromatography (HPLC) has been the method of choice (3, 4, 5), few methods developed to date have provided reliable qualitative and quantitative information when analyzing just a few picomoles (pmol) of PTH amino acid. The most common problems in PTH analysis have been baseline fluctuations due to gradient elution, the lack of resolution of all PTH amino acids from each other and from frequently observed by-products of sequencing such as N, N’-diphenylthiourea (DPTU) and the elution of basic residues such as PTH-arginine in broad peaks which are difficult to quantify.