scholarly journals Immunofluorescence of histone H1 in stimulated lymphocytes measured by flow cytophotometry.

1979 ◽  
Vol 27 (1) ◽  
pp. 417-425 ◽  
Author(s):  
J W Betz ◽  
C J Herman
Keyword(s):  
Regulation Of Gene Expression ◽  
Fluorescein Isothiocyanate ◽  
Bimodal Distribution ◽  
Cross Reactivity ◽  
Peripheral Lymphocytes ◽  
Microcomplement Fixation ◽  
Flow Cytophotometry ◽  
High Fluorescence ◽  
Rna Complexes ◽  
Number Of Cells

The modification of histones or their redistribution during the transition from actively transcribing chromatin to the heterochromatic chromosomes seems to play a major in regulation of gene expression. The purpose of this study was to monitor the change in immunofluorescence of histone HI during phytohemagglutinin stimulation in peripheral lymphocytes. The histone antigens were prepared from pig thymus, proven to be pure by gel electrophoresis and repeatedly injected as RNA-complexes into rabbits. The antihistone HI antiserum titer was 1:4000, and there was no cross-reactivity with other histone fractions as shown by microcomplement fixation tests. Affinity chromatography purified antibody after being labeled with fluorescein isothiocyanate was able to differentially stain HeLa cells as controls and those, where histone HI had been extracted by perchloric acid treatment. The measurements were done on a Los Alamos Scientific Laboratories-flow cytophotometer cell sorter. Staining peripheral lymphocytes resulted in a bimodal distribution. The increase in number of cells with high fluorescence intensity had its maximum about 20 hr before the maximum proliferative activity of the lymphocytes as measured by number of cells in S phase with the DNA-stain mithramycin.

scholarly journals Determination of Morphine in Human Urine by the Novel Competitive Fluorescence Immunoassay

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Jie Cao ◽  
Xiao-Ying Chen ◽  
Wu-Rong Zhao
Keyword(s):  
Monoclonal Antibodies ◽  
Human Urine ◽  
Screening Tool ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
The Novel ◽  
Fluorescence Immunoassay ◽  
Coated Plate ◽  
Efficient Screening

A competitive fluorescence immunoassay for the identification and quantification of morphine has been developed on the basis of hapten-coated plate format. Hapten was prepared through covalent conjugating a morphine derivative with albumin bovine. In the immunoassay, the hapten was inoculated on a 96-well plate and then bound with monoclonal antibodies labeled with a signal indicating dye, fluorescein isothiocyanate (FITC). Unbound FITC-antibodies were rinsed off from the plate. The fluorescein intensity decreases in the presence of morphine molecules due to the competitively binding to antibodies against hapten. The intensity is inversely correlated with the concentration of morphine. In quantitative analysis for urine samples, we obtained a linearity range of 0.2 μg/mL∼2.5 μg/mL, along with a detection limit of c.a. 1 ng/mL. The fluorescence immunoassay shows low cross-reactivity (below 10%) to 6-acetylmorphine, 3-acetylmorphine, and heroine. The developed method produced comparable results to the standard GC-MS/MS method. In conclusion, a rapid and efficient screening tool for morphine in clinical human urine has been established.

scholarly journals Flow-cytometric analysis of chicken red blood cells.

1978 ◽  
Vol 26 (3) ◽  
pp. 170-186 ◽  
Author(s):  
D Bloch ◽  
N Beaty ◽  
C T Fu ◽  
E Chin ◽  
J Smith ◽  
...  
Keyword(s):  
Sodium Dodecyl ◽  
Flow Cytometric Analysis ◽  
Fluorescence Emission ◽  
Bimodal Distribution ◽  
Parameter Analysis ◽  
Main Peak ◽  
Acceptance Angle ◽  
Flow Cytometric ◽  
High Fluorescence ◽  
Chicken Erythrocytes

Flow-cytometric analysis of acriflavin-Feulgen stained chicken erythrocytes shows a complex distribution of amounts of deoxyribonucleic acid fluorescence, the profile consisting of a main peak and a right hand shoulder. This bimodal distribution, an artifact characteristically seen on analysis of flattened cells using orthogonal flow systems, results from fluorescence emission in preferred directions stemming from the combined effects of refractility and orientation of the cells. The shoulder disappears on analysis of lysed erythrocyte ghosts, also on analysis of cells in a medium whose refractive index approximates that the cells. An orientation effect for matrue erythrocytes was indicated by reanalysis of fractions after sorting on the basis of high and low fluorescence or scatter signals. Both fractions gave the original range of values on reanalysis, although some changes in shape of the profile and in the peak positions for the sorted cells were seen. Sodium dodecyl sulfate treatment of stained cells "loosened" the cells' structure, yielding lowered scatter values, and fluorescence values approaching those of the shoulder. The average fluorescence emission of the erythrocytes was lower than that of reticulocytes and lymphocytes. The values of the latter correspond closely, although coincidently, to that the erythrocyte shoulder values. Dual parameter analysis of forward light scatter, and fluorescence, which was detected at 90 degrees to the laser beam, showed the low fluorescence to be accompanied by low scatter signal, and the high fluorescence among the cells with the high scatter signal. The lowered forward scatter signal is due to a wider scattering of light from cells oriented edge-on to the detector, and loss of signal beyond the acceptance angle of the detector. These results suggest that the preferred directions for fluorescence are in the plane of the cells, and the values are dependent on the cells' orientation in the stream. These interpretations were supported by the results of analysis of partially oriented cells. The approaches used and conclusions arrived at are similar to those of Gledhill et al (16), Van Dilla et al (37), in their analysis of fluorescence of flat sperm cells although the affects in the case of the erythrocytes are less extreme.

The fluorescent antibody test for the identification of strains of Clostridium botulinum type E

1970 ◽  
Vol 16 (10) ◽  
pp. 917-921 ◽  
Author(s):  
A. C. Emeruwa ◽  
F. E. Ashton ◽  
R. Z. Hawirko
Keyword(s):  
Fluorescent Antibody ◽  
Fluorescein Isothiocyanate ◽  
Antibody Test ◽  
Cross Reactivity ◽  
Specific Staining ◽  
Vegetative Cells ◽  
Carbonate Buffer ◽  
Clostridium Botulinum Type E ◽  
Homologous Strain ◽  
The Cross

A fluorescent antibody test for the identification of toxigenic and nontoxigenic type E strains of C. botulinum is described. Hyperimmune rabbit sera were prepared to cell suspensions of the D8, 070, and Beluga strains and to spores of the D8 and PM-15 strains. Fluorescein isothiocyanate immunoglobulin conjugates were prepared and absorbed with C. bifermentans and diluted to eliminate non-specific staining and cross-reactivity with related serotypes. Enzyme pretreatment of spores and washing the stained spores in carbonate buffer, pH 9.0, enhanced the sensitivity of the test. Conjugates of cell antiserum, in most cases, stained the spore bodies and not the vegetative cells of the cross-reacting strains. Conjugates of spore antiserum stained vegetative cells as well as spores but the fluorescence of the cells was eliminated by absorption of the conjugates with young cells of the homologous strain. The absorbed conjugates stained spores of 19/20 isolates from diverse geographical areas without any reduction in the intensity of fluorescence, indicating that spore antigens carry the determinant groups of type E specificity and that the common antigen is a component of the spore body. In addition, some of the cross-reacting strains showed the same degree of fluorescence as the homologous strain, suggesting that more than one antigen may be shared by these strains.

scholarly journals Presence of plasma cells binding autologous antibody during an immune response.

1979 ◽  
Vol 150 (5) ◽  
pp. 1265-1270 ◽  
Author(s):  
S Jackson ◽  
J Mestecky
Keyword(s):  
Immune Response ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Plasma Cells ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Lymphoid Tissues ◽  
Specific Antibodies ◽  
Tetramethylrhodamine Isothiocyanate ◽  
Idiotypic Antibody

Spleen and other lymphoid tissues of rabbits immunized with human serum albumin (HSA) and human lactoferrin (LF) were examined for the presence of cells forming anti-idiotype antibodies. To detect these cells, IgG, F(ab')2, or Fab' of specific antibodies were isolated, fluorochrome-tagged with tetramethylrhodamine isothiocyanate, and used as an idiotypic marker to detect splenic plasma cells that are producing anti-idiotypic antibody. By this procedure, we were able to demonstrate anti-idiotypic cells in surprisingly high numbers. For example, in six rabbits immunized with HSA for periods ranging from 36 to 542 d, the percentage of Ig-positive cells that stained with autologous idiotype ranged from 0.7 to 44; furthermore, cross-reactivity was observed among seven different anti-HSA preparations and two anti-LF antisera. The isotype of anti-idiotypic cells, determined by costaining with fluorescein isothiocyanate-labeled goat Fc-specific anti-rabbit Ig, was shown to be predominantly IgG. These findings provide evidence of the presence of plasma cells producing antibody to autologous idiotype during a vigorous immune response.

Preliminary investigation of the use of indirect immunofluorescence to detect conidia of Brunchorstiapinea

1989 ◽  
Vol 19 (3) ◽  
pp. 389-392 ◽  
Author(s):  
Carol L. Nelson ◽  
John D. Castello ◽  
Paul D. Manion
Keyword(s):  
New York ◽  
Indirect Immunofluorescence ◽  
Preliminary Investigation ◽  
New York State ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Staining Procedure ◽  
Spore Trap ◽  
Indirect Immunofluorescence Staining

An indirect immunofluorescence staining procedure was developed for detection of Brunchorstiapinea conidia, using antiserum to conidia and a commercially prepared fluorescein isothiocyanate protein A conjugate. The procedure detected B. pinea conidia from culture, pycnidia, and spore-trap collections. Although cross reactivity occurred with spores of Fusarium spp., Sirococcus sp., Phialophora sp., Gliocladium sp., Verticillium sp., and Gelatinosporium sp., these spores were easily distinguished from those of B. pinea by size, shape, septation, and degree of fluorescence. Fluorescent B. pinea like conidia were collected in spore traps located within and outside the New York State quarantine region. However, the identity of B. pinea like conidia could not be corroborated by other methods.

scholarly journals Development of a Monoclonal Antibody Specific to the IgM Heavy Chain of Bighead Catfish (Clarias macrocephalus): A Biomolecular Tool for the Detection and Quantification of IgM Molecules and IgM+ Cells in Clarias Catfish

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 567
Author(s):  
Anurak Bunnoy ◽  
Uthairat Na-Nakorn ◽  
Prapansak Srisapoome
Keyword(s):  
Heavy Chain ◽  
Clarias Gariepinus ◽  
Rabbit Model ◽  
Molecular Data ◽  
Cross Reactivity ◽  
African Catfish ◽  
Sequence Information ◽  
Blood Leukocytes ◽  
Clarias Macrocephalus ◽  
Flow Cytophotometry

Catfish is a commonly-cultivated freshwater fish in Thailand and many Southeast Asian countries. The molecular data obtained for the IgM heavy chain (IgMH) of catfish have been useful for distinguishing monoclonal antibodies (mAbs). A mAb specific to Cμ1 of the IgMH of catfish (IgMHCμ1 mAb) was developed in a rabbit model using sequence information from bighead catfish (Clarias macrocephalus). The IgMHCμ1 mAb strongly recognized the IgM heavy chain of the tested catfish, namely, bighead catfish, African catfish (Clarias gariepinus) and their hybrid (C. macrocephalus × C. gariepinus), in immunological Western blot analysis and competitive ELISAs. Additionally, the IgMHCμ1 mAb successfully recognized IgM+ cells by detecting IgM molecules in both secreted and membrane-bound forms in peripheral blood leukocytes (PBLs). The IgMHCμ1 mAb was further used to quantify the percentage of IgM+ cells among PBLs through flow cytophotometry. The IgM+ cell percentages of healthy bighead catfish, African catfish and their hybrid were 38.0–39.9%, 45.6–53.2%, and 58.7–60.0%, respectively. Furthermore, the IgMHCμ1 mAb showed no cross-reactivity with the IgM of zebrafish. These findings suggest that this mAb can be used as an immunological tool for monitoring the health, immune status, and immune development of cultivated Clarias catfish.

scholarly journals Development and Preliminary Application of Multiplex Loop-Mediated Isothermal Amplification Coupled With Lateral Flow Biosensor for Detection of Mycobacterium tuberculosis Complex

2021 ◽  
Vol 11 ◽  
Author(s):  
Xingyun Wang ◽  
Guirong Wang ◽  
Yacui Wang ◽  
Shuting Quan ◽  
Hui Qi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Tuberculosis Complex ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Tuberculosis Complex ◽  
Loop Mediated Isothermal Amplification

The aim of this study was to develop a simple and reliable method to detect Mycobacterium tuberculosis complex (MTBC) and verify its clinical application preliminarily. A loop-mediated isothermal amplification method coupled with lateral flow biosensor (LAMP-LFB) assay, was developed and evaluated for detection of MTBC. Two sets of primers, which targeted IS6110 and IS1081 sequences of MTBC, were designed for establishment of multiplex LAMP-LFB assay. The amplicons were labelled with biotin and fluorescein isothiocyanate (FITC) by adding FITC labelled primer and biotin-14-dATP and biotin-14-dCTP and could be visualized using LFB. The optimal reaction conditions of multiplex LAMP-LFB assay confirmed were 66°C for 50 min. The analytical sensitivity of multiplex LAMP-LFB is 10 fg of genomic templates using pure culture, and no cross-reactivity with other common bacteria and non-tuberculous mycobacteria strains was obtained. A total of 143 clinical samples collected from 100 TB patients (62 definite TB cases and 38 probable TB cases) and 43 non-TB patients were used for evaluating the feasibility of multiplex LAMP-LFB assay. The multiplex LAMP-LFB (82.0%, 82/100) showed higher sensitivity than culture (47.0%, 47/100, P < 0.001) and Xpert MTB/RIF (54.0%, 54/100, P < 0.001). Importantly, the multiplex LAMP-LFB assay detected additional 28 probable TB cases, which increased the percentage of definite TB cases from 62.0% (62/100) to 90.0% (90/100). The specificity of multiplex LAMP-LFB assay in patients without TB was 97.7% (42/43). Therefore, multiplex LAMP-LFB assay is a simple, reliable, and sensitive method for MTBC detection, especially in probable TB cases and resource limited settings.

Monitoring the insecticidal toxins fromBacillus thuringiensisin soil with flow cytometry

1997 ◽  
Vol 43 (11) ◽  
pp. 1074-1078 ◽  
Author(s):  
H. Tapp ◽  
G. Stotzky
Keyword(s):  
Flow Cytometry ◽  
Bacillus Thuringiensis ◽  
Internal Control ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Significant Shift ◽  
Dot Blot ◽  
Natural Habitats ◽  
Insecticidal Toxins ◽  
The Right

The accumulation and persistance in soil and other natural habitats of the insecticidal toxins from Bacillus thuringiensis may result in environmental hazards, such as toxicity to nontarget species and the selection of toxin-resistant target species. We describe the use of flow cytometry as a method for detecting and tracking the fate of these insecticidal toxins in soil that does not require their extraction and purification. The toxins from B. thuringiensis subspp. tenebrionis and kurstaki were bound on clay- or silt-sized particles separated from Kitchawan soil that was unamended (naturally contains predominantly kaolinite) or amended to 6% v/v with the clay minerals montmorillonite or kaolinite (as an internal control). The particle–toxin mixtures were suspended in 0.1 M phosphate buffer (pH 7) containing 3% nonfat milk powder to block nonspecific binding of antibody, resuspended in a solution of antibody to the toxin from B. thuringiensis subsp. tenebrionis, and then resuspended in a solution of anti-rabbit antibody conjugated with fluorescein isothiocyanate (FITC–Ab). Controls consisted of the particles alone and bound complexes of the particles with the toxin from B. thuringiensis subsp. kurstaki. All particles that bound the toxin from B. thuringiensis subsp. tenebrionis showed a significant shift in the peak of fluorescence to the right on the x axis as compared with the nonspecific fluorescence from the control FITC–Ab complexes with particles in the absence of the toxin. There was also a slight shift in the peak to the right for some particles that bound the toxin from B. thuringiensis subsp. tenebrionis, as there is some cross-reactivity between the toxins from B. thuringiensis subspp. tenebrionis and kurstaki and the antibodies that they induce. This method is more sensitive and rapid than the dot-blot ELISA, and processing of many samples is easily accomplished.Key words: flow cytometry, soil, insecticidal toxins, Bacillus thuringiensis, clay, silt.

scholarly journals Detection of Cryptosporidium parvum oocysts in calf fecal samples by direct immunofluorescence assay

2011 ◽  
Vol 20 (4) ◽  
pp. 269-273 ◽  
Author(s):  
Weslen Fabricio Pires Teixeira ◽  
Willian Marinho Dourado Coelho ◽  
Cáris Maroni Nunes ◽  
Marcelo Vasconcelos Meireles
Keyword(s):  
Cryptosporidium Parvum ◽  
Polyclonal Antibodies ◽  
Deae Cellulose ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunofluorescence ◽  
Fecal Samples ◽  
Direct Immunofluorescence Assay

The aim of this study was to produce a conjugate containing anti-Cryptosporidium parvum polyclonal antibodies and standardize a Direct Immunofluorescence Assay (DIF) for detecting C. parvum oocysts in fecal samples from calves. In order to obtain anti-C. parvum polyclonal antibodies, two New Zealand rabbits were immunized with a purified solution of C. parvum oocysts and Freund's adjuvant. Purification of the immunoglobulin G (IgG) fraction was performed by means of precipitation in ammonium sulfate and chromatography using a DEAE-cellulose column. The anti-C. parvum polyclonal antibody titer was determined by means of the enzyme-linked immunosorbent assay (ELISA). The rabbit anti-C. parvum IgG fraction was conjugated with fluorescein isothiocyanate and standardization of the DIF was performed using various dilutions of conjugate on slides positive for C. parvum oocysts. The cross-reactivity of the anti-C. parvum conjugate was tested using oocysts of Cryptosporidium serpentis, Cryptosporidium andersoni, Escherichia coli, Eimeria sp., and Candida sp. An anti-C. parvum conjugate was successfully produced, thus allowing standardization of DIF for detection of Cryptosporidium oocysts in fecal samples. Cross-reactivity of anti-C. parvum polyclonal antibodies with C. andersoni and C. serpentis was also observed.

scholarly journals STUDIES ON ALKALINE AND ACID RIBONUCLEASES IN MAMMALIAN TISSUES IMMUNOHISTOCHEMICAL LOCALIZATION AND IMMUNOCHEMICAL PROPERTIES

1967 ◽  
Vol 15 (11) ◽  
pp. 662-673 ◽  
Author(s):  
SHIGERU MORIKAWA
Keyword(s):  
Small Intestine ◽  
Fluorescent Antibody ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Immunohistochemical Localization ◽  
Rnase A ◽  
Specific Antibodies ◽  
Mammalian Tissues ◽  
Tetramethylrhodamine Isothiocyanate ◽  
Bovine Pancreas

The distribution of alkaline and acid ribonucleases (RNases) was investigated in bovine tissues using fluorescent antibody techniques. The immunochemical properties of the bovine RNases were also examined. Specific antibodies against acid and alkaline RNase obtained by immunizing rabbits with the corresponding antigens were conjugated with fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate. Anti-alkaline RNase antibody reacted against alkaline RNase-A, and anti-acid RNase antibody reacted against two different acid RNases with no cross-reactivity between alkaline and acid RNases. Phosphate-buffered 10% formalin was the best general fixative among those examined, but some differences in staining results were observed depending upon the tissue and fixative employed. Two different patterns of distribution of alkaline RNase in pancreas and kidney were observed depending upon the fixative employed. The localization of RNases in bovine pancreas, liver, small intestine, kidney, spleen, lymph node, thymus, adrenal and heart were investigated and the physiologic roles of the RNases were discussed.

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