Purified histoplasmin reagents for the identification of h and m antigens and homologous antibodies by immunodiffusion or complement fixation tests.
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Antibodies, Fungal↗
Biomedical subjects
W K Harrell
Publications and source records attributed to W K Harrell.
Identification of Salmonella O antigens by coagglutination.
This study concerns the preparation of reagents for identifying the somatic O antigens of Salmonella enteritidis. Coagglutination reagents (COAGs) with antibody fixed to killed and stabilized protein A-bearing staphylococci were prepared with antisera which were used for identifying the somatic O antigens of S. enteritidis by the slide agglutination test. The reactions of the COAGs were compared with those obtained with the grouping antisera in routine slide agglutination tests in which 41 or more serologically different Salmonella strains, representing most of the known groups, were used. One-third of the COAGs gave identical reactions to those of the slide agglutination antisera. The reactions of the other COAGs varied from the slide agglutination antisera results, some by many reactions and others by only a few. The coagglutination procedure was more reactive than the routine slide agglutination test and resulted in cross-reactions which were not observed in the original grouping antisera. More COAGs were specific when they were tested with alcohol-treated cultures than with live cultures. Coagglutination conserves antiserum, allowing about 12 times as many tests for a given volume of group-specific glycerolized antiserum as does the slide agglutination method.
Agglutination Tests↗
Factors influencing the reactivity of Legionella antigens in immunofluorescence tests.
We examined several factors for their effects on the serological reactivity of Legionella antigens used for direct or indirect fluorescent-antibody tests. These factors included media, methods of killing, strain differences, and the nature of the reactivity with diverse human sera. The maximum serological reactivities were obtained with charcoal-yeast extract agar; the relative antigenicity of cells grown on a chemically defined medium could be fourfold less than those grown on the charcoal-yeast extract agar. Cells grown at 25 degrees C showed only small antigenic differences from those grown at 35 degrees C but had better morphological and staining characteristics. Cells killed by 1% Formalin or 37% Formalin vapors showed a 20% less relative antigenicity than those killed by heat, but their cell walls stained more clearly and they had fewer aberrations. As tested with several human sera, cells of Philadelphia 1 showed great variation in relative antigenicity with changes in media or methods of preparation; Bellingham 1 was quite stable under these same conditions. The data suggest that Bellingham 1 had serogroup 1-specific antigens, reactive with human sera, which were not present in Philadelphia 1.
Antigens, Bacterial↗
Evaluation of candidate international reference reagents and a microimmunodiffusion test for the identification of precipitins to the H and M antigens of histoplasmin.
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Antibodies, Fungal↗
Metal requirements of Legionella pneumophila.
Serial passage of six strains of Legionella pneumophila and one strain of Pseudomonas aeruginosa in a liquid chemically defined medium deficient in trace metals resulted in the death of five L. pneumophila strains and very limited growth in the remaining strain and the P. aeruginosa strain. Addition of either iron or magnesium restored growth to almost normal levels in all of the strains when early-passage inocula were used. A low concentration of magnesium stimulated growth with cobalt, copper, iron, manganese, molybdenum, vanadium, or zinc. When a complete defined medium containing trace metals was used, growth was inhibited by adding the chelators ethylenediaminetetraacetic acid, citrate, or 2,2'-bipyridyl. Chelator inhibition was partly or fully relieved with either calcium, cobalt, copper, iron, magnesium, molybdenum, nickel, vanadium, or zinc. P. aeruginosa differed from L. pneumophila in that it required higher concentrations of each chelator to inhibit growth and that its growth was stimulated by only four metals: calcium, iron, magnesium, and zinc. A trace-metal supplement for L. pneumophila was designed which included all metals stimulating growth in these experiments and which proved to be sufficient for optimal growth of all the strains.
Amino Acids↗
Improved streptococcal grouping antisera containing polyethylene glycol.
Antisera to streptococcal groups A through G containing 4% polyethylene glycol 6000 were prepared and evaluated. Seventy strains of homologous and heterologous beta- and non-beta-hemolytic streptococci were included in the evaluation. Homologous reactions were determined against extracts prepared by four extraction methods: hot hydrochloric acid (Lancefield) extraction, autoclave extraction, hot formamide extraction, and nitrous acid extraction. Enhancement of the precipitin reaction in the presence of polyethylene glycol 6000 permitted a fourfold dilution of all antisera for use in the capillary precipitin test. At this dilution, the potency of the antisera exceeded requirements established for these reagents.
Cross Reactions↗
Amino acid requirements of Legionella pneumophila.
The amino acids required for growth and as energy sources by 10 strains of Legionella pneumophila were determined by using a chemically defined medium. All strains required arginine, cysteine, isoleucine, leucine, threonine, valine, methionine, and phenylalanine or tyrosine. Most strains (7 of 10) required serine, and two strains had to be supplied proline before growth could be established. All 10 strains used serine and, to a lesser extent, threonine as the sole sources of carbon and energy. The Y serine calculated was 94.9 +/- 8.5 g (dry weight) of cells/mol of serine. Assuming that the value of Y adenosine 5'-triphosphate is 10.5, these results indicate that oxidative catabolism of 1 mol of serine yielded approximately 9 mol of adenosine 5'-triphosphate. This high yield suggests that although serine was the major source of carbon, other amino acids may also be metabolized.
Amino Acids↗
Preparation of reference antisera for laboratory diagnosis of blastomycosis.
Antiserum was prepared in rabbits against the A antigen and a new antigen (labeled the D antigen) of yeast-phase cells of Blastomyces dermatitidis by using immunoelectrophoretic precipitin arcs as vaccine. The antiserum can be used as a reference serum in the immunodiffusion test for identyfying antibodies to the A antigen in sera from patients thought to have blastomycosis. Antibodies to the D antigen have not yet been found in human sera. The rabbit antiserum that was absorbed with Histoplasma capsulatum yeast-phase cells did not react in the complement fixation test with either H. capsulatum yeast-phase or mycelial-phase complement fixation antigen. Therefore, it could also be used as a positive control serum in the complement fixation test. A fluorescent-antibody conjugate prepared from the absorbed serum was found to be valuable for the identification of yeast-phase cells of B. dermatitidis.
Animals↗
Development of a chemically defined liquid medium for growth of Legionella pneumophila.
A chemically defined liquid medium has been developed for the study of the physiology and antigen production of the Legionnaires disease bacterium. The medium contains basal salts, vitamins, alpha-ketoglutaric acid, pyruvate, 0.05% l-cysteine, 0.05% glutathione, and a mixture of 20 additional amino acids, each of 0.01% final concentration, except serine, which was at 0.1%. The medium in shake culture at 37 degrees C with increased CO2 at pH 6.5, supports the maximum rate of growth, the highest cell yields, and the maximum cell surface antigen as distinguished by specific fluorescein isothiocyanate-conjugated antibody. Studies during the development of this medium showed that CO2, pyruvate, and alpha-ketoglutarate strongly stimulated growth; that cysteine and methionine were required for growth; and that serine, threonine, histidine, tyrosine, and tryptophane were energy sources. Glutathione substituted for cysteine, but cystine did not. The organisms did not use glucose and polysaccharides, as judged by cell yields when these carbohydrates were present or absent. The chelators malate, citrate, and ethylenediaminetetraacetic acid totally inhibited growth. Beta-mercaptoethanol, thioglycolate, dithiothreitol, and Tween 80 (0.05%) inhibited growth strongly or completely. Catalase activity was extremely weak or absent. Morphology varied, depending upon conditions and phases of growth. In general, filamentous forms became chains of cigar-shaped bacilli fragmenting to pairs and becoming coccoidal in the late stationary pha-e of growth. The organism grew at 25, 30, and 37 degrees C. Although they varied in their growth characteristics, 10 isolates were passed for five transfers in the chemically defined broth, giving maximum rates of growth, cell yields, and antigen production.
Aerobiosis↗
Immunogenicity and characteristics of M protein released by phage-associated lysin from group-A streptococci types 1 and 23.
A phage-associated lysin (PAL) was used to release M protein from goup-A streptococci of types 1 and 23. Much of the lysin-released-M protein (LYSIN-M) of both types was of high molecular weight, since LYSIN-M appeared just after the void volume on Sephadex G-200 gel-filtration. Some of the LYSIN-M appeared just after the void volume on Sephadex G-200 gel-filtration. Some of the LYSIN-M of both types was found to be firmly attached to group-A carbohydrate. Type-1 LYSIN-M was partially purified by ammonium-sulfate precipitation followed by absorption and elution from an immunoabsorbent column containing antibody for group-A carbohydrate. Type-23 LYSIN-M was partially purified by precipitation at its isoelectric point, pH 4-9. Rabbits immunised in the footpads with either type-1 or type-23 LYSIN-M responded by producing both precipitins and bactericidal (opsonising) antobodies. Some of the antiesera were absorbed and rendered specific for homologous acid extracts. The LYSIN-M preparations of both types 1 and 23 were originally contaminated with heat-labile antigen(s). Antibodies to these heat-labile antigen(s), which cross-react from type to type, were found in the type-specific antisera distributed by the Center for Disease Control. The specificity of Lancefield typing antisera depends on their being tested with extracts of streptococci prepared at pH 2 and 100 degree C for 10 min. Although LYSIN-M is more difficult to prepare and purify then acid-heat released M protein, it might prove useful for studying the nature of native streptococcal M protein.
Antigens, Bacterial↗
H and M antigens of Histoplasma capsulatum: preparation of antisera and location of these antigens in yeast-phase cells.
Antiserum has been prepared in rabbits against the H and M antigens of H. capsulatum with immunoelectrophoretic precipitin arcs used as vaccines. The antiserum is specific for H. capsulatum in the immunodiffusion test and can be used as reference serum for identifying antibodies to these antigens in sera from suspected cases histoplasmosis. We found that (1) hand m antigens are not located on the surface of yeast-phase cells and (ii) complement fixation releases the antigens reactive in the complement fixation test from yeast-phase cells.
Antibodies, Fungal↗
Stability of fluorescent antibody conjugates stored under various conditions.
Two experiments were carried out to determine the stability of fluorescent antibody conjugates. In experiment 1, Francisella tularemia conjugates in the lyophilized state retained their original staining titer for 1,294 days when stored at 25, 4 to 5, and -20 C; at 37 C the conjugates were stable for at least 65 days. In the liquid state at pH 7.4 and 8.0 these conjugates were stable for 1,294 days at 4 to 5 and -20 C, whereas those stored at 25 C remained stable through days 473 and 160 of storage, respectively, after which the staining titer gradually dropped. In experiment 2 five previously lyophilized conjugates were rehydrated with three different diluents and stored at 4 to 5 C for up to 600 days at their working dilutions. All of these conjugates retained their original staining titer during the test period except an anti-human globulin conjugate rehydrated with phosphate-buffered saline. Recommendations are made for the long-term storage of fluorescent antibody conjugates.
Antibodies, Anti-Idiotypic↗
Simple procedure for production by group C streptococci of phage-associated lysin active against group A streptococci.
Phage-associated lysin of high potency was prepared by growing the host group C streptococcal strain 26RP66 in a semisynthetic medium. The lysin was stabilized by adding dithiothreitol and neutralized ethylenediaminetetraacetic acid (EDTA) to facilitate further concentration and partial purification. The lysin remained active when stored at -65 C for 1 year. Lysin was active against all strains of group A streptococci tested and was more active against living cells than heat-killed cells. The procedure outlined is practicable for most bacteriological research laboratories and does not require column purification or other complex biochemical procedures. It should be useful to any laboratory which requires small amounts of lysin to produce L-forms and protoplasts or to release streptococcal antigens.
Ammonium Sulfate↗
Evaluation of histoplasmin for the presence of H and M antigens: some difficulties encountered in the production and evaluation of a product suitable for the immunodiffusion test.
The development of a single histoplasmin suitable for use in immunodiffusion testing of a variety of human histoplasmosis sera depends not only upon the presence of both antigens, but on relative concentration of one to the other, and perhaps on the chemical characteristics of the H and M antigens of a given strain.
Antigens, Fungal↗
Reuse of Salmonella and Shigella absorbing cells for preparing monospecific Salmonella O and Shigella antisera.
Salmonella and Shigella organisms used as absorbing cells for preparing group-or type-specific Salmonella O or Shigella antisera may be reused four or more times without qualitatively reducing their capacity to remove undesired antibodies. The cells may be reclaimed by heating in flowing steam or by treatment with N/5 HCl. The former method is preferred.
Absorption↗
Quantitative measurement of precipitating antibodies and standardization of Streptococcus group B antisera by the reverse radial immunodiffusion technique.
A comparative study was made of the reverse radial immunodiffusion (RRID) technique and the quantitative precipitin test for determining the amount of precipitable antibodies present in streptococcus group B antisera. The coefficient of correlation between the 2 tests was 0.963 when a purified carbohydrate antigen was used. The results of this study show that: (1) a group B antiserum should contain at least 3 mg of precipitable antibodies per ml to ensure that it will react with acid extracts of group B diagnostic cultures; and (2) the RRID technique is an accurate and simple procedure for determining the amount of precipitable antibody present in an antiserum, and it could be used for the standardization of these diagnostic reagents. A method for preparing the group B carbohydrate antigen used in the RRID and quantitative precipitin tests is described.
Animals↗
Quantitative measurement of precipitating antibodies in streptococcal grouping antisera by the single radial immunodiffusion technique.
A comparative study was made of the single radial immunodiffusion test and the classical quantitative precipitin test for determining the amount of precipitable antibodies present in streptococcal groups A and C antisera. The potency of 21 group A and 54 group C antisera was determined by both methods; purified group-specific carbohydrates were used as antigens. The coefficient of correlation between the results from the two methods was 0.976 for group A antisera and 0.946 for group C antisera. When the concentration of antigen, the volume of antiserum used, and the depth of the antigen-agar mixture are kept constant, the diameter of the precipitin disc is directly related to the concentration of precipitable antibodies present in the antiserum. The use of the radial immunodiffusion test for evaluating and standardizing streptococcal grouping antisera is discussed as well as the advantages and disadvantages of using a concentrated vaccine for producing these antisera.
Agar↗
Cross-protective antigens of group A streptococci types 3 and 31 and types 46 and 51.
Reciprocal cross-protective antigens have been demonstrated between types 3 and 31 cocci, and one-way cross-protective antigens have been demonstrated between types 46 and 51 cocci. The reciprocal cross-protective antigen of types 3 and 31 is distinct from the specific M protein of either type. In the one-way cross relationship, type 46 cocci contain both type 46 and type 51 M proteins and therefore stimulate protective antibodies against both types. Type 51 cultures contain only the homologous M antigen. These relationships were demonstrated by capillary precipitin tests, indirect bactericidal tests, and in agar-gel diffusion patterns. The practical significance of these relationships in the serological typing of group A streptococci is discussed along with their possible role in immunity to streptococcal infections in man.
Animals↗