Joseph F. Metzger

Immune Electron Microscopy

Summary A method is described by which immune systems can be studied at subcellular level with the electron microscope, using a ferritin-antibody conjugate. A conjugated immune serum to Staphylococcus aureus was applied to homologous organisms, embedded in methacrylate with uranium and thin sections cut. An antigen-antibody reaction was detected by attachment of the ferritin molecule in the capsule-like material. Use of formalin in primary fixation for electron microscopy as well as nonspecific immunofluorescence is discussed.

Microheterogeneity of staphylococcal enterotoxin B

Abstract Four components have been demonstrated in staphylococcal enterotoxin B by isoelectric focusing in polyacrylamide gels. In a typical preparation their relative concentrations from the most to least cathodic were 6, 56, 31 and 7%. The components were not stable conformers nor were the differences in isoionic points due to bound ligand. A comparison of the measured isoionic points with values calculated for varying numbers of excess basic groups indicated that each component differed from the succeeding one by a single charge. Sequential conversion of the components from the more to the less alkaline forms was obtained by exposure to pH 9.0 at 37 °C. The data gave an excellent fit for two consecutive first-order reacions in whicc the specific reaction rate constants were nearly identical. The reaction was much slower at pH 7.3 and 37 °C and no change at all was observed over a period of 32 days at pH 9.0 and 1 °C. Amide hydrolysis is the mechanism for these conversions, but the rate is too slow to account for the appearance of four components during the comparatively short period of bacterial fermentation. It is suggested, therfore, that only the most cathodic component is synthesized by the organism and that this is converted to the other forms enzymatically.

Stabilization of native structure by the closed disulfide loop of staphylococcal enterotoxin B

Abstract The extent and rate of reduction of aqueous staphylococcal enterotoxin B by dithiothreitol in the absence or presence of the denaturant guanidine hydrochloride were directly proportional to the extent and rate of enterotoxin B unfolding. Thus, the single disulfide bond of native enterotoxin B must be positioned in a region of tertiary structure. Reduction followed by alkylation with iodoacetamide or iodoacetic acid of enterotoxin B unfolded in 8 M urea resulted, after refolding in dilute alkaline buffer, in aggregation of the alkylated monomer with a significant increase in viscosity. Also, the stability of alkylated enterotoxin B toward denaturation by guanidine hydrochloride was greatly diminished as compared to unmodified enterotoxin B. However, the refolded, alkylated enterotoxin derivatives retained the ability to completely precipitate rabbit antibody specific for native toxin. Tryptic hydrolysis of a single peptide bond in the 92–112 disulfide loop also significantly lowered the stability of the protein to guanidine denaturation. It is proposed, therefore, that the closed disulfide loop of enterotoxin B stabilizes a minor domain of structure whose disruption increases the conformational flexibility of the toxin molecule without extensive denaturation and loss of biologic specificity.

Fractionation and purification of Staphylococcus aureus enterotoxin B by electrofocusing

Abstract Electrofocusing fractionation of purified Staphylococcus enterotoxin B was performed utilizing pH 7–9, 7–10, and 9.0–9.5 ampholine—sucrose gradients. On the pH 7–10 focusing experiments, four physically distinct, but antigenically similar entities were isolated. The major component (isoionic point pH 9.4 at 4°C) was refocused and found to be stable.

Identification of Pathogenic Fungi in Surgical and Autopsy Specimens by Immunofluorescence.

A method is presented for the preparation of immune sera and detection by immunofluorescence ofC. immitis, S. schenckii, B. dermatitidis, C. neoformans, andC. albicans in surgical and autopsy material. Formalin fixation does not affect the antigens of the mycotic agents. There are no cross reactions except withC. immitis andC. neoformans, which can be differentiated by the site of the specific fluorescence in each organism.A method is presented for the preparation of immune sera and detection by immunofluorescence ofC. immitis, S. schenckii, B. dermatitidis, C. neoformans, andC. albicans in surgical and autopsy material. Formalin fixation does not affect the antigens of the mycotic agents. There are no cross reactions except withC. immitis andC. neoformans, which can be differentiated by the site of the specific fluorescence in each organism.

Effect of chloromycetin and streptomycin on embryonic tissue growth in in vitro tissue culture.

Summary Growth of normal embryonic tissue was completely inhibited by concentrations of 90000 and greater μg/ml of streptomycin. Growth was partially inhibited by concentrations of 480 μg/ml through the highest concentration of Chloromycetin tested.

The pH dependence of enterotoxin polymerization by formaldehyde.

Abstract Toxoiding of staphylococcal enterotoxin B is accomplished by polymerizing the exotoxin into large, water-soluble antigen complexes in neutral or acid formaldehyde solution (Warren, J. R., Spero, L. and Metzger, J. F. (1973) J. Immunol. 111, 885). Experimental data for the presumptive nature of the amino acid sites in the enterotoxin B molecule at which intermolecular cross-linking by formaldehyde occurs are now reported. The polymerization of enterotoxin B in aqueous formaldehyde buffered at pH 7.5 was almost completely inhibited by added lysine; polymerization of enterotoxin at pH 5.0 was inhibited by added lysine or N-acetyltyrosine. Small model compounds for arginine, asparagine, glutamine, histidine and tryptophan residues were without effect. These results suggest that enterotoxin was polymerized at neutral pH by cross-linking between lysine residues, at acid pH by cross-linking between pairs of lysine and tyrosine residues. Thus, a high degree of chemical specificity and a marked pH dependence of this specificity are the major features of enterotoxin polymerization by formaldehyde.

Demonstration of Venezuelan equine encephalomyelitis in tissue culture by immunofluorescence.

Summary In a guinea pig heart cell line, infected with an egg-adapted strain of Trinidad VEE, the virus could be demonstrated by immunofluorescence prior to development of CPE. Formalin fixation preserved the antigenicity, and the cover slips could be stored in this fixative at 4°C for periods up to 2 weeks without diminution of specific fluorescence. A whole burro serum conjugated with fluorescein isothiocyanate with a counterstain added proved a more reliable reagent than fractionated or absorbed sera. The model as described has the possibility of obtaining a specific viral diagnosis at the clinical level in the period normally required for initial isolation by conventional cell culture technics.

Phage typing of antibiotic-resistant staphylococci. III, Infection, cross-infection and superinfection.

IN previous reports1 , 2 the high incidence of similar phage patterns of the antibiotic-resistant staphylococci that were isolated from lesions from various clinical sources within a large medical ...

Serologic typing of Listeria monocytogenes by gel diffusion using thermostable antigens.

Summary A procedure is described by which acid extracts of capsularenhanced L. monocytogenes are serotyped by gel diffusion. Sixty strains were placed in 5 serotypes that coincide with the serotypes obtained by conventional methods using somatic “O” and flagellar “H” factor sera. The procedure has been simplified so that it can be efficiently utilized by facilities other than specialized typing centers. We are indebted to Dr. Milton M. Gray for confirming serotypes of cultures and advice during this investigation.