Physicochemical Characterization of Direct Fluorescent Antibody Reagents

Hébert, Guillaume; Pittman, Bertie; Cherry, William B.

doi:10.1177/002203457605500119011

Cited by 22 publications

(28 citation statements)

References 3 publications

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“…Cells were serotyped with florescent antibody prepared by one of us (B.K.) by using previously described procedures (7,11,12), except that Sepharose 4B beads rather than Sepharose 6MB beads were used in the affinity column (7). The following antisera were used: A .…”

Section: Methodsmentioning

confidence: 99%

Actinomyces georgiae sp. nov., Actinomyces gerencseriae sp. nov., Designation of Two Genospecies of Actinomyces naeslundii, and Inclusion of A. naeslundii serotypes II and III and Actinomyces viscosus serotype II in A. naeslundii Genospecies 2

Johnson

Moore²,

Kaneko³

et al. 1990

International Journal of Systematic Bacteriology

153

132

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Section: Methodsmentioning

confidence: 99%

Actinomyces georgiae sp. nov., Actinomyces gerencseriae sp. nov., Designation of Two Genospecies of Actinomyces naeslundii, and Inclusion of A. naeslundii serotypes II and III and Actinomyces viscosus serotype II in A. naeslundii Genospecies 2

Johnson

Moore²,

Kaneko³

et al. 1990

International Journal of Systematic Bacteriology

153

132

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“…Microscopic agglutination titers were approximately 1:6000. The immunoglobulin fraction was obtained by three cycles of precipitation with 35% saturated ammonium sulfate and redissolving the resultant precipitate in water (8). The final fraction was desalted by chromatography through a Sephadex G-25 column using saline as the eluent and stored at -70°C (9).…”

mentioning

confidence: 99%

Movement of antibody-coated latex beads attached to the spirochete Leptospira interrogans.

Charon¹,

Lawrence²,

O’Brien³

1981

Proc. Natl. Acad. Sci. U.S.A.

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Antibody-coated latex beads (Ab-beads) were attached to Leptospira interrogans serovars iMini 3055 and icterohaemorrhagiae SC1157. The movement of the Ab-beads relative to the motion of the cells was observed by direct darkfield microscopy or was recorded on videotape. When the Ab-beads were attached to the front end of motile cells, the Ab-beads were displaced towards the back end of the cells. When the cells reversed direction, the Ab-beads also reversed direction. A number of hypotheses were proposed and tested to account for this Ab-bead displacement. The one best supported by the evidence states that the Ab-beads are attached to antigens of the outer membrane sheath. These antigens are dragged laterally through the sheath due to the forward motion of the cells and the retarding forces of the medium acting on the beads. The results obtained provide information on the nature of the outer membrane sheath of L. interrogans, the basis for certain movements of spirochetes, and insight on how spirochetes attach to eukaryotic cells and tissues. In addition, the results indicate that antigens can move laterally through membranes as rapidly as 11 ,tm/sec. Leptospira interrogans is a spirochete with a well-characterized ultrastructure and a unique mode of motility. Outermost is an outer membrane sheath (OS), and within this sheath is the righthanded helical cell cylinder and two axial filaments (AF) (1-4). Each AF is subterminally attached to the cell cylinder, and genetic evidence suggests that the AFs are directly involved in motility (5). On the basis of this and other evidence, a motility model was proposed which states that rotational motors at the base of the AFs propel the organisms in a manner analogous to the flagella of rod-shaped bacteria (6).In the course of testing this motility model, we coated latex beads with antibodies directed to whole cells (Ab-beads) and attached these Ab-beads to swimming cells. Our goal was to track the rotational movement ofthe Ab-beads as the cells swam in a given direction. However, we were surprised to find that when Ab-beads were attached to the front end ofthe cells they were rapidly displaced along the length ofthe cells until the Abbeads reached the back end. The Ab-beads reversed direction as the cells reversed direction. These and related motions of cells and the attached Ab-beads were recorded on videotape, and tracings from these tapes are presented in this communication. The results suggest that the Ab-beads are attached to antigens ofthe OS. These antigens are dragged laterally through the OS to the back end of the cells due to the forward motion of the cells and the retarding forces of the medium acting on the beads. EXPERIMENTAL PROCEDURES Bacteria. L. interrogans serovar illini 3055 (referred to as serovar illini), and the linear motility mutants DB115, DB218, DB290, and DB340 derived from serovar illini have been described (5). These linear mutants lack the hook-and spiralshaped ends typical of wild-type serovar illini; they are also deficient in both t...

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“…Fluorescein/protein (F/P) ratios of IgG-FITC conjugates were determined from A ratios (A494/A280), which were then related to F/P ratios as determined by the biuret analysis for protein and A494 for FITC (17). F/P ratios of IgG conjugates were within the range of 14 to 20 /pg/mg.…”

Section: Methodsmentioning

confidence: 99%

Diethylaminoethyl-cellulose-bacterial cell immunoadsorbent columns: preparation of serotype-specific globulin and immunofluorescent conjugates for Streptococcus mutans serotypes a and d

McKinney¹,

Thacker²

1976

Infect Immun

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Diethylaminoethyl (DEAE)-cellulose was used as a support material for preparing bacterial cell columns. Pretreatment of the bacterial cells with formalin was essential in obtaining satisfactory adherence of the cells to DEAE-cellulose. Cross-reacting antibodies were removed from antibody preparations against strains of Streptococcus mutans serotypes a and d by adsorption on appropriate bacterial cell columns. S. mutans serotype d was further divided into two subtypes on the basis of immunofluorescent staining with conjugates of immunospecifically adsorbed immunoglobulin G. The DEAE-cellulose-bacterial cell columns were regenerated after use by desorbing the cross-reacting antibodies with low-pH buffer and were used repeatedly over an 18-month period with no detectable loss in effectiveness. ethyl ether, 1 meq/g) was from Eastman Kodak Co., Rochester, N.Y. The pH 7.4 borate saline (BS) was prepared as 0.05 M boric acid containing 0.85% NaCl. The pH was adjusted to 7.4 with concentrated NaOH, and the conductivity was adjusted to 15,000 ,umhos/cm at room temperature by adding water or granular NaCl. Conductivity was measured with a Beckman conductivity bridge model RC 16B2 (Beckman Instruments, Inc., Cedar Grove, N.J.). The pH 8.0 BS was prepared as 0.05 M boric acid containing 1161 on August 1, 2020 by guest http://iai.asm.org/ Downloaded from

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Physicochemical Characterization of Direct Fluorescent Antibody Reagents

Cited by 22 publications

References 3 publications

Actinomyces georgiae sp. nov., Actinomyces gerencseriae sp. nov., Designation of Two Genospecies of Actinomyces naeslundii, and Inclusion of A. naeslundii serotypes II and III and Actinomyces viscosus serotype II in A. naeslundii Genospecies 2

Actinomyces georgiae sp. nov., Actinomyces gerencseriae sp. nov., Designation of Two Genospecies of Actinomyces naeslundii, and Inclusion of A. naeslundii serotypes II and III and Actinomyces viscosus serotype II in A. naeslundii Genospecies 2

Movement of antibody-coated latex beads attached to the spirochete Leptospira interrogans.

Diethylaminoethyl-cellulose-bacterial cell immunoadsorbent columns: preparation of serotype-specific globulin and immunofluorescent conjugates for Streptococcus mutans serotypes a and d

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