scholarly journals Use of monoclonal and polyclonal antibodies against DNA adducts for the detection of DNA lesions in isolated DNA and in single cells.

1985 ◽  
Vol 62 ◽  
pp. 81-88 ◽  
Author(s):  
R A Baan ◽  
O B Zaalberg ◽  
A M Fichtinger-Schepman ◽  
M A Muysken-Schoen ◽  
M J Lansbergen ◽  
...  
Keyword(s):  
Dna Adducts ◽  
Polyclonal Antibodies ◽  
Single Cells ◽  
Dna Lesions ◽  
Antibodies Against Dna

scholarly journals Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies.

1986 ◽  
Vol 34 (3) ◽  
pp. 287-292 ◽  
Author(s):  
S Van Noorden ◽  
M C Stuart ◽  
A Cheung ◽  
E F Adams ◽  
J M Polak
Keyword(s):  
Alkaline Phosphatase ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Single Cells ◽  
Pituitary Hormones ◽  
Tissue Binding ◽  
Human Pituitary ◽  
Single Section ◽  
End Products ◽  
Species Specific

Mouse monoclonal and rabbit polyclonal antibodies to human pituitary hormones were applied together to sections of normal and neoplastic human pituitary tissue. Binding sites were revealed with species-specific immune reagents combined with various enzymes (peroxidase, alkaline phosphatase, and beta-D-galactosidase). The enzymes were developed separately to give differently colored end-products. Where two hormones were present in the same cell, a mixed color was produced. Up to four hormones could be immunostained in a single section. Multiple immunoenzymatic staining has great potential for the analysis of plural antigen production by single cells and relationships between cells producing different antigens.

The clonal analysis of anticardiolipin antibodies in a single patient with primary antiphospholipid syndrome reveals an extreme antibody heterogeneity

Blood ◽  
2001 ◽  
Vol 97 (12) ◽  
pp. 3820-3828 ◽  
Author(s):  
Patricia Lieby ◽  
Anne Soley ◽  
Honey Levallois ◽  
Benedicte Hugel ◽  
Jean-Marie Freyssinet ◽  
...  
Keyword(s):  
Antiphospholipid Syndrome ◽  
Antiphospholipid Antibodies ◽  
Polyclonal Antibodies ◽  
Single Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Clonal Analysis ◽  
Primary Antiphospholipid Syndrome ◽  
Single Patient ◽  
Prothrombotic State

The mechanism underlying the prothrombotic state that characterizes the primary antiphospholipid syndrome proves to be difficult to define mainly because of the variety of the phospholipid and protein targets of antiphospholipid antibodies that have been described. Much of the debate is related to the use of polyclonal antibodies during the different antiphospholipid assays. To better describe the antiphospholipid antibodies, a strategy was designed to analyze the reactivity of each one antibody making up the polyclonal anticardiolipin activity, breaking down this reactivity at the clonal level. This was performed in a single patient with primary antiphospholipid syndrome by combining (1) the antigen-specific selection of single cells sorted by flow cytometry using structurally bilayered labeled anionic phospholipids and (2) the cloning of immunoglobulin (Ig) variable (V) region genes originating from individual IgG anticardiolipin-specific B cells by a single-cell polymerase chain reaction technique. The corresponding V regions were cloned in order to express human recombinant antibodies in insect cells by a baculovirus expression system. The molecular analysis, the fine specificity, and the protein cofactor dependency of the first 5 monoclonal IgG anticardiolipins are reported here. This clonal analysis reveals the extreme heterogeneity of these antibodies, which could account for the difficulties in the previous attempts to define the pathogenic antiphospholipid response. This approach should help to unravel the complex antiphospholipid immune response and the mechanism of the prothrombotic state associated with these antibodies, but it could also shed some light on their possible origins.

Use of polyclonal antibodies against carcinogen–DNA adducts in analysis of carcinogenesis

1998 ◽  
Vol 102-103 ◽  
pp. 441-446 ◽  
Author(s):  
Tomoyuki Shirai ◽  
Satoru Takahashi ◽  
Lin Cui ◽  
Yasuyuki Yamada ◽  
Mariko Tada ◽  
...  
Keyword(s):  
Dna Adducts ◽  
Polyclonal Antibodies

Biological monitoring exposure of workers from plant producing carbon electrodes: quantification of benzo[a]pyrene DNA adducts in leukocytes, by a 32P-postlabelling method and an immunoassay

1999 ◽  
Vol 18 (5) ◽  
pp. 314-321 ◽  
Author(s):  
J P Arnould ◽  
A Pfohl-Leszkowicz ◽  
V Bach ◽  
J P Libert ◽  
J Belegaud
Keyword(s):  
Dna Adducts ◽  
Aromatic Hydrocarbons ◽  
Human Exposure ◽  
Polyclonal Antibodies ◽  
Carbon Electrodes ◽  
Competitive Immunoassay ◽  
High Exposure ◽  
Potential Indicator ◽  
Polycyclic Aromatic ◽  
Good Detection

The levels of benzo[a]pyrene were monitored for blood DNA-benzo[a]pyrene adducts in 17 workers from a plant producing carbon electrodes, with high exposure to benzo[a]pyrene (575-902-1149 ng m-3). Two different techniques, a 32P-postlabelling method and a competitive immunoassay using polyclonal antibodies obtained from rabbits immunised with DNA modified by benzo[a]pyrenetrans-7,8-dihydrodiol-9,10-epoxide were used. For each worker, urinary 1-hydroxypyrene, a potential indicator of exposure to polycyclic aromatic hydrocarbons, was measured. The effect of tobacco by urinary cotinine measurement was also considered. The postlabelling and immunoassay detection limits for DNA-benzo[a]pyrene adducts were respectively 0.15 and 10 fmol 50 ug-1 of DNA. The results obtained by the two methods demonstrated a good detection of DNA-benzo[a]pyrene adducts, but no direct relationship between the quantity of adducts and the concentration of benzo[a]pyrene in air-borne was noted in the studied plant. The levels of DNA-benzo[a]pyrene adducts obtained by immunoassay were significantly higher than those obtained by the 32P-postlabelling (P50.001). For several workers, variations due to professional or non professional factors must be taken into account in interpreting the results. In conclusion, the two methods used proved very efficient in determining DNA-benzo[a]pyrene adducts, and may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.

scholarly journals Detection of Streptococcus Suis by in Situ Hybridization, Indirect Immunofluorescence, and Peroxidase-Antiperoxidase Assays in Formalin-Fixed, Paraffin-Embedded Tissue Sections from Pigs

2000 ◽  
Vol 12 (3) ◽  
pp. 224-232 ◽  
Author(s):  
Mette Boye ◽  
Anne A. Feenstra ◽  
Conny Tegtmeier ◽  
Lars Ole Andresen ◽  
Søren R. Rasmussen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Indirect Immunofluorescence ◽  
Polyclonal Antibodies ◽  
Single Cells ◽  
Streptococcus Suis ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1–31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

scholarly journals STRIDE—a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells

2019 ◽  
Vol 48 (3) ◽  
pp. e14-e14 ◽  
Author(s):  
Magdalena M Kordon ◽  
Mirosław Zarębski ◽  
Kamil Solarczyk ◽  
Hanhui Ma ◽  
Thoru Pederson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Cells ◽  
Dna Lesions ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dna Breaks ◽  
Damage Site ◽  
Double Strand Dna Breaks ◽  
In Situ Detection ◽  
Double Strand Dna

Abstract We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. The sensitivity of STRIDE was tested using a specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used terminal deoxynucleotidyl transferase dUTP nick-end labeling assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions) and fragmentation of DNA in human spermatozoa. The STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

scholarly journals Simultaneous detection of multiple DNA damage types by multi-colour fluorescent labelling

2019 ◽  
Vol 55 (76) ◽  
pp. 11414-11417 ◽  
Author(s):  
Dmitry Torchinsky ◽  
Yael Michaeli ◽  
Natalie R. Gassman ◽  
Yuval Ebenstein
Keyword(s):  
Dna Damage ◽  
Dna Adducts ◽  
Single Molecule ◽  
Simultaneous Detection ◽  
Dna Lesions ◽  
Major Obstacle ◽  
Fluorescent Labelling ◽  
Dna Molecule ◽  
Repair Enzymes ◽  
Damage Types

Specific and simultaneous quantitation of DNA adducts is a major obstacle. Using repair enzymes, we present a protocol to quantify two types of DNA lesions simultaneously on the same DNA molecule and examine repair dynamics by single-molecule imaging.

Detection of Carcinogen–Macromolecular Adducts in Humans

1989 ◽  
Vol 8 (5) ◽  
pp. 913-932 ◽  
Author(s):  
A. Weston ◽  
D. K. Manchester ◽  
A. Povey ◽  
C. C. Harris
Keyword(s):  
Dna Adducts ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Target Cells ◽  
High Exposure ◽  
Increased Risk ◽  
Polycyclic Aromatic ◽  
Human Sera ◽  
Carcinogen Exposure ◽  
Risk Of Cancer

A major concern of molecular epidemiology is the identification of individuals at increased risk of cancer by obtaining evidence of high exposure to carcinogens that may lead to pathobiological lesions in target cells. DNA is considered to be a target for modification by mutagens and carcinogens; therefore, damage to DNA can be used as an internal, molecular dosimeter of carcinogen exposure. The reactive species of these carcinogens may bind either directly to DNA to form adducts or indirectly to cause secondary DNA lesions through free radicals and aldehydes. Highly sensitive and specific methods have been developed to measure DNA lesions and DNA repair products that are found in biological specimens from humans exposed to carcinogens in the environment. For example, DNA adducts have been measured in cells and tissues from people exposed environmentally to carcinogenic polycyclic aromatic hydrocarbons or alkylating agents. Antibodies recognizing carcinogen-DNA adducts have also been detected in human sera. Carcinogen-protein adducts are also being used as molecular dosimeters of carcinogen exposure. The advantages and limitations of the various methods used to measure carcinogen-macromolecular adducts are discussed here. The use of two or more complementary assays to obtain confirmatory results is recommended.

scholarly journals HMGB1 bound to cisplatin–DNA adducts undergoes extensive acetylation and phosphorylation in vivo

2015 ◽  
Vol 6 (3) ◽  
pp. 2074-2078 ◽  
Author(s):  
Yafeng He ◽  
Yin Ding ◽  
Dan Wang ◽  
Wanjun Zhang ◽  
Weizhong Chen ◽  
...  
Keyword(s):  
Dna Adducts ◽  
Dna Lesions ◽  
Novel Method

Here, an application of a biomolecular probe based on a peptide–oligonucleotide conjugate is presented as a novel method for investigating the recognition of cisplatin–DNA lesions by HMGB1 in vivo.

scholarly journals Direct, sensitive and specific detection of individual single- or double-strand DNA breaks by fluorescence microscopy

2019 ◽  
Author(s):  
Magdalena Kordon ◽  
Mirosław Zarębski ◽  
Kamil Solarczyk ◽  
Hanhui Ma ◽  
Thoru Pederson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fluorescence Microscopy ◽  
Single Cells ◽  
Primary Cultures ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Damage Site ◽  
Double Strand Dna Breaks ◽  
Age Related ◽  
Double Strand Dna

ABSTRACTWe here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. Sensitivity of STRIDE was tested using specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used TUNEL assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions), and fragmentation of DNA in human spermatozoa. STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

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