scholarly journals STUDIES ON FLUORESCENT ANTIBODY STAINING

1961 ◽  
Vol 114 (1) ◽  
pp. 89-110 ◽  
Author(s):  
Gerald Goldstein ◽  
Irene S. Slizys ◽  
Merrill W. Chase
Keyword(s):  
Fluorescent Antibody ◽  
Deae Cellulose ◽  
Fluorescein Isothiocyanate ◽  
Gradient Elution ◽  
Fluorescent Staining ◽  
Coupling Ratio ◽  
Specific Fluorescence ◽  
Antibody Staining ◽  
Fluorescent Products ◽  
Elution Chromatography

1. A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2. In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3. Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: (a) conjugating only gamma globulins with fluorescein isothiocyanate; (b) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; (c) use of crystalline preparations of fluorescein isothiocyanate. 4. Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5. The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6. A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules.

scholarly journals ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS

1971 ◽  
Vol 49 (2) ◽  
pp. 390-404 ◽  
Author(s):  
George M. Maniatis ◽  
Vernon M. Ingram
Keyword(s):  
Experimental Error ◽  
Single Cells ◽  
Deae Cellulose ◽  
Fluorescein Isothiocyanate ◽  
Erythroid Cells ◽  
Fluorescent Staining ◽  
Ammonium Sulfate Precipitation ◽  
Blood Smears ◽  
Tetramethylrhodamine Isothiocyanate ◽  
Cellulose Column

Rabbit antibodies specific for the major tadpole and frog hemoglobin components of R. catesbeiana were used for the detection of the two hemoglobins inside single cells. The antisera, after fractionation by ammonium sulfate precipitation and diethylaminoethyl (DEAE)-cellulose chromatography, were conjugated with fluorescein isothiocyanate for the antifrog hemoglobin serum and tetramethylrhodamine isothiocyanate for the antitadpole hemoglobin serum. The conjugated fractions, refractionated by stepwise elution from a DEAE-cellulose column, were used for the fluorescent staining of blood smears, liver tissue imprints, and smears of liver cell suspensions. Both simultaneous and sequential staining with the two fluorescent preparations indicated that larval and adult hemoglobins were not present within the same erythrocyte during metamorphosis. In other experiments, erythroid cells from animals in metamorphosis were spread on agar containing specific antiserum. Precipitates were formed around the cells which contain the particular hemoglobin. The percentages of cells containing either tadpole or frog hemoglobin were estimated within the experimental error of the method. The data showed that the two hemoglobins are in different cells. It is concluded that the hemoglobin change observed during the metamorphosis of R. catesbeiana is due to the appearance of a new population of erythroid cells containing exclusively frog hemoglobin.

scholarly journals Physical, immunochemical, and functional properties of Acanthamoeba profilin.

1984 ◽  
Vol 98 (1) ◽  
pp. 214-221 ◽  
Author(s):  
P C Tseng ◽  
M S Runge ◽  
J A Cooper ◽  
R C Williams ◽  
T D Pollard
Keyword(s):  
Fluorescent Antibody ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Sedimentation Equilibrium ◽  
Binding Assays ◽  
Cytoplasmic Matrix ◽  
Unbound Fraction ◽  
Antibody Staining ◽  
High Concentrations ◽  
Equilibrium Ultracentrifugation

Acanthamoebe profilin has a native molecular weight of 11,700 as measured by sedimentation equilibrium ultracentrifugation and an extinction coefficient at 280 nm of 1.4 X 10(4) M-1cm-1. Rabbit antibodies against Acanthamoeba profilin react only with the 11,700 Mr polypeptide among all other ameba polypeptides separated by electrophoresis. These antibodies react with a 11,700 Mr polypeptide in Physarum but not with any proteins of Dictyostelium or Naeglaria. Antibody-binding assays indicate that approximately 2% of the ameba protein is profilin and that the concentration of profilin is approximately 100 mumol/liter cells. During ion exchange chromatography of soluble extracts of Acanthamoeba on DEAE-cellulose, the immunoreactive profilin splits into two fractions: an unbound fraction previously identified by Reichstein and Korn (1979, J. Biol. Chem., 254:6174-6179) and a tightly bound fraction. Purified profilin from the two fractions is identical by all criteria tested. The tightly bound fraction is likely to be attached indirectly to the DEAE, perhaps by association with actin. By fluorescent antibody staining, profilin is distributed uniformly throughout the cytoplasmic matrix of Acanthamoeba. In 50 mM KCl, high concentrations of Acanthamoeba profilin inhibit the elongation rate of muscle actin filaments measured directly by electron microscopy, but the effect is minimal in KCl with 2 MgCl2. By using the fluorescence change of pyrene-labeled Acanthamoeba actin to assay for polymerization, we confirmed our earlier observation (Tseng, P. C.-H., and T. D. Pollard, 1982, J. Cell Biol. 94:213-218) that Acanthamoeba profilin inhibits nucleation much more strongly than elongation under physiological conditions.

scholarly journals Description of a polyvalent conjugate and a new serogroup of Bacteroides melaninogenicus by fluorescent antibody staining

1976 ◽  
Vol 3 (5) ◽  
pp. 506-512
Author(s):  
D W Lambe ◽  
R C Jerris
Keyword(s):  
Fluorescent Antibody ◽  
Fluorescein Isothiocyanate ◽  
Rapid Identification ◽  
Antibody Staining ◽  
Biochemical Testing ◽  
Fluorescent Antibody Staining

A polyvalent conjugate (fluorescein isothiocyanate-labeled antibody reagent) containing serogroups A, B, and C conjugates was prepared. This polyvalent conjugate gave a positive fluorescent antibody (FA) stain with 49 stains of Bacteroides melaninogenicus representing serogroups A, B, and C. When additional strains (92 strains) of the three subspecies of B. melaninogenicus were examined by the FA stain, with A, B, and C, and polyvalent conjugates, nine strains of B. melaninogenicus subsp. intermedius failed to give a positive stain with any conjugate. Therefore, an FA conjugate was prepared with the antiserum to one of these strains (532-70A); all nine strains stained positively with this conjugate. These nine strains were biochemically characteristic of B. melaninogenicus subsp. intermedius; thus, these strains were designated as a new serogroup, serogroup C-1. A new polyvalent conjugate containing serogroups A, B, C, and C-1 was prepared. This polyvalent conjugate stained positively with 23 representative strains from serogroups A, B, C, and C-1. The new conjugates failed to stain positively with other anaerobes and aerobes tested. The four individual conjugates, as well as the polyvalent conjugate, may be used for a more rapid identification of B. melaninogenicus than is possible by biochemical testing.

scholarly journals Immunohistochemical detection of Clostridia species in paraffin-embedded tissues of experimentally inoculated guinea pigs

2005 ◽  
Vol 25 (1) ◽  
pp. 4-8 ◽  
Author(s):  
Ronnie A. Assis ◽  
Francisco C.F. Lobato ◽  
Rogéria Serakides ◽  
Renato L. Santos ◽  
Guilherme R.C. Dias ◽  
...  
Keyword(s):  
Guinea Pigs ◽  
Fluorescent Antibody ◽  
Deae Cellulose ◽  
Fluorescein Isothiocyanate ◽  
Type A ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Blackleg is caused by Clostridium chauvoei, whereas malignant oedema is caused by C. chauvoei, C. septicum, C. sordellii, C. perfringens type A, and/or C. novyi type A. Anti-C. chauvoei, anti-C. septicum, anti-C. sordellii and anti-C. novyi type A polyclonal antibodies were produced in rabbits and purified in a column of DEAE-cellulose. Aliquots of the antisera were conjugated with fluorescein isothiocyanate and the remaining was used for the streptavidin biotin peroxidase technique (SBPT). SBPT was standardized to detect C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs. SBPT was compared to a fluorescent antibody technique (FAT). Sections and smears of muscle from inoculation area (MIA), heart, liver, spleen and kidney, were obtained for both SBPT and FAT. Cross-reactions between the different Clostridial species were not observed. C. chauvoei and C. septicum were detected in all specimens from the animals inoculated with these microorganisms, while only sections of muscle obtained from all the animals inoculated with C. sordellii and C. novyi type A were positive. The same results observed by the SBPT, were obtained on tissue smears of these microorganisms stained by the FAT. The results indicate that SBPT is suitable for detection of C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs.

scholarly journals LOCALIZATION OF CARBONIC ANHYDRASE IN AVIAN GASTRIC MUCOSA, SHELL GLAND AND BONE BY IMMUNOHISTOCHEMISTRY

1974 ◽  
Vol 22 (8) ◽  
pp. 819-825 ◽  
Author(s):  
CAROL V. GAY ◽  
HARALD SCHRAER ◽  
EDWARD J. FALESKI ◽  
ROSEMARY SCHRAER
Keyword(s):  
Carbonic Anhydrase ◽  
Fluorescent Antibody ◽  
Fluorescein Isothiocyanate ◽  
Shell Gland ◽  
Significant Finding ◽  
Specific Fluorescence ◽  
Tubular Gland ◽  
Γ Globulins ◽  
Mucosal Lining ◽  
Avian Erythrocyte

Antisera to purified avian erythrocyte carbonic anhydrase (CA) were produced in rabbits and the γ-globulin fractions were isolated and purified by ammonium sulfate precipitation and diethylaminoethyl-Sephadex chromatography. The anti-CA immuno-γ-globulins were tested by immunoelectrophoresis and judged to be highly specific for CA. Fluorescein isothiocyanate-conjugated goat antirabbit γ-globulins were used in the indirect fluorescent antibody localization of CA in cryostat sections. Specific fluorescence of CA was observed in gastric mucosal lining cells, shell gland columnar and tubular gland cells, red blood cells, erythroblasts and osteoclasts. Specific fluorescence was absent in the several immunologic controls, including the blocking test. Specific fluorescence was also lacking in tissue constituents which contain no detectable amounts of CA, i.e., muscle, connective tissue, blood vessels and nuclei. A significant finding in this study is the occurrence of cross-reactions between antibodies to red blood cell CA and CA from other tissues, indicating the immunologic similarity of CA from different tissue sources.

FLUORESCENT ANTIBODY STAINING IN THE SERODIAGNOSIS OF TRICHINOSIS

1963 ◽  
Vol 9 (4) ◽  
pp. 625-628 ◽  
Author(s):  
R. K. Baratawidjaja ◽  
Ann Hewson ◽  
N. A. Labzoffsky
Keyword(s):  
Complement Fixation ◽  
Trichinella Spiralis ◽  
Fluorescent Antibody ◽  
Immunofluorescent Staining ◽  
Staining Procedure ◽  
Fluorescent Staining ◽  
Antibody Staining ◽  
Human Sera ◽  
Fluorescent Antibody Staining

A fluorescent staining procedure for Trichinella spiralis and the appearance of the stained larvae are described. The applicability of the method to the sero-diagnosis of trichinosis was investigated. The results obtained both with the experimental and human sera agreed well with the complement-fixation results. In titrating 9 experimental sera and 36 sera from parasitologically proved or clinically diagnosed cases of trichinosis in humans, higher titers were obtained by the immunofluorescent staining, indicating that this test is somewhat more sensitive.

Characterization of Bacteroides gingivalis by direct fluorescent antibody staining and cellular fatty acid profiles

1982 ◽  
Vol 28 (4) ◽  
pp. 367-374 ◽  
Author(s):  
Dwight W. Lambe Jr. ◽  
Kaethe P. Ferguson ◽  
William R. Mayberry
Keyword(s):  
Fatty Acid ◽  
Fluorescent Antibody ◽  
Fluorescein Isothiocyanate ◽  
Cellular Fatty Acid ◽  
Fatty Acid Profiles ◽  
Bacteroides Species ◽  
Antibody Staining ◽  
Direct Fluorescent Antibody ◽  
Bacteroides Gingivalis ◽  
Fluorescent Antibody Staining

Bacteroides gingivalis is a newly proposed species which includes strains isolated from the mouth. Thirteen strains of B. gingivalis isolated from three geographic locations in the United States and France were examined with direct fluorescent antibody staining and analysis of total cellular fatty acids and compared with 16 strains of B. asaccharolyticus of nonoral origin by the same methods. Bacteroides gingivalis strains reacted with the B. gingivalis conjugate (fluorescein isothiocyanate labeled antibody reagent) only, while the B. asaccharolyticus strains reacted with the B. asaccharolyticus conjugate only. The B. gingivalis strains showed negative fluorescence with fluorescein isothiocyanate conjugates for other black-pigmented Bacteroides species. The specificity of the B. gingivalis conjugate was demonstrated by its failure to stain 88 strains of aerobic and anaerobic bacteria other than B. gingivalis. The fatty acid profiles of B. gingivalis and B. asaccharolyticus were readily distinguishable. The B. gingivalis profile was also distinguishable from those of other pigmenting Bacteroides species on the basis of concentration ratios among the characteristic components. These results support the species separation of B. gingivalis and B. asaccharolyticus. Further, they indicate the usefulness of cellular fatty acid profiles as an adjunct to the use of specific fluorescent antibody conjugates for identification of Bacteroides species.

scholarly journals Studies on Oxidized Wool IV. Fractionation of Proteins Extracted From Wool on Deae-Cellulose Using Buffers Containing 8m Urea

1961 ◽  
Vol 14 (3) ◽  
pp. 461 ◽  
Author(s):  
IJ O'donnell ◽  
EOP Thompson
Keyword(s):  
Deae Cellulose ◽  
Gradient Elution ◽  
Soluble Proteins ◽  
Elution Curves ◽  
Elution Chromatography ◽  
Gradient Elution Chromatography

aand y-keratoses, which are soluble proteins extracted from oxidized wool, give widely spread zones in their elution curves when examined by gradient elution chromatography on columns of diethylaminoethyl (DEAE)-cellulose in buffers containing 8M urea. Stepwise elution of the proteins indicates that they could consist of many components.

scholarly journals Mycoplasmas in cell cultures from rheumatoid synovial membranes

1971 ◽  
Vol 69 (1) ◽  
pp. 17-25 ◽  
Author(s):  
K. B. Fraser ◽  
P. V. Shirodaria ◽  
Margaret Haire ◽  
D. Middleton
Keyword(s):  
Rheumatoid Arthritis ◽  
Tissue Culture ◽  
Fluorescent Antibody ◽  
Fluorescent Staining ◽  
Antibody Staining ◽  
Synovial Fluids ◽  
Direct Fluorescent Antibody ◽  
The Common ◽  
Synovial Membranes ◽  
Fluorescent Antibody Staining

SUMMARYTen strains of myocoplasmas were recovered from cultures of synovium or cultures inoculated with synovial fragments from rheumatoid arthritis and one from osteo-arthritis. The source of the organisms is not known. Patients with rheumatoid arthritis had no complement-fixing antibody and no fluorescent staining antibody against the mycoplasmas isolated and no mycoplasma antigen was detected by immunofluorescence in sections of synovia and in synovial fluids.The strains isolated were of two main serological types and could be distinguished by direct fluorescent antibody staining from standard types of human commensals and the common tissue-culture contaminants. One may beMycoplasma laidlawii.

A Most Probable Number Method for the Enumeration of Legionella Bacteria in Water

1991 ◽  
Vol 24 (2) ◽  
pp. 143-147 ◽  
Author(s):  
N. A. Grabow ◽  
R. Kfir ◽  
W. O. K. Grabow
Keyword(s):  
Water Samples ◽  
Membrane Filtration ◽  
Quantitative Method ◽  
Fluorescent Antibody ◽  
Most Probable Number ◽  
Probable Number ◽  
Comparative Tests ◽  
Antibody Staining ◽  
Direct Fluorescent Antibody ◽  
Yeast Extract Agar

A new quantitative method for the enumeration of Legionella bacteria in water is described. Appropriate tenfold serial dilutions of water samples concentrated by membrane filtration are plated in triplicate on buffered charcoal yeast extract agar. After incubation for 3 days representative smears from individual plates are tested for the presence of Legionella by direct fluorescent antibody staining. The number of positive plates in each dilution is used to calculate the Legionella count by means of conventional most probable number statistics. In comparative tests on a variety of water samples this method yielded significantly higher counts than previously used procedures.

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