scholarly journals Enzyme-linked immunosorbent assays for Z-DNA

1988 ◽  
Vol 255 (1) ◽  
pp. 53-59 ◽  
Author(s):  
M J Thomas ◽  
J S Strobl
Keyword(s):  
Alkaline Phosphatase ◽  
Horseradish Peroxidase ◽  
Polyclonal Antiserum ◽  
Dot Blot ◽  
Base Pairs ◽  
Rabbit Polyclonal Antiserum ◽  
Sperm Dna ◽  
Immunosorbent Assays ◽  
Z Dna ◽  
Plasmid Puc8

Dot blot and transblot enzyme-linked immunosorbent assays (e.l.i.s.a.) are described which provide sensitive non-radioactive methods for screening Z-DNA-specific antisera and for detecting Z-DNA in polydeoxyribonucleotides and supercoiled plasmids. In the alkaline phosphatase dot blot e.l.i.s.a., Z-DNA, Br-poly(dG-dC).poly(dG-dC), or B-DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), Br-poly(dI-dC).poly(dI-dC), or salmon sperm DNA were spotted onto nitrocellulose discs and baked. The e.l.i.s.a. was conducted in 48-well culture dishes at 37 degrees C using a rabbit polyclonal antiserum developed against Br-poly(dG-dC).poly(dG-dC), an alkaline phosphatase-conjugated second antibody, and p-nitrophenol as the substrate. Under conditions where antibody concentrations were not limiting, alkaline phosphatase activity was linear for 2 h. Dot blot e.l.i.s.a. conditions are described which allow quantification of Z-DNA [Br-poly(dG-dC).poly(dG-dC)] within the range 5-250 ng. Dot blot and transblot horseradish peroxidase e.l.i.s.a. are described that detect Z-DNA within supercoiled plasmid DNAs immobilized on diazophenylthioether (DPT) paper. In the transblot e.l.i.s.a., plasmid pUC8 derivatives containing 16, 24, or 32 residues of Z-DNA were electrophoresed in agarose gels and electrophoretically transferred to DPT paper. Z-DNA-antibody complexes were detected by the horseradish peroxidase-catalysed conversion of 4-chloro-1-naphthol to a coloured product that was covalently bound to the DPT paper. Z-DNA antibody reactivity was specific for supercoiled Z-DNA containing plasmids after removal of the antibodies cross-reactive with B-DNA by absorption onto native DNA-cellulose. The transblot e.l.i.s.a. was sensitive enough to detect 16 base pairs of alternating G-C residues in 100 ng of pUC8 DNA.

scholarly journals Assessment of a method for immunochemical detection of antigen on nitrocellulose membranes.

1989 ◽  
Vol 37 (2) ◽  
pp. 257-263 ◽  
Author(s):  
N M Chu ◽  
A J Janckila ◽  
J H Wallace ◽  
L T Yam
Keyword(s):  
Alkaline Phosphatase ◽  
Horseradish Peroxidase ◽  
Reaction Medium ◽  
Intestinal Alkaline Phosphatase ◽  
Dot Blot ◽  
Western Blots ◽  
Calf Intestinal Alkaline Phosphatase ◽  
Immunochemical Detection ◽  
Staining Methods ◽  
Secondary Antibodies

Immunoblotting techniques are widely used for detection of antigen immobilized on nitrocellulose membranes. There are many immunolabeling methods and staining methods available to disclose the presence of antigen in such techniques. Five common staining methods each for alkaline phosphatase and horseradish peroxidase were examined. The staining methods with the highest sensitivity and the lowest background were selected for studies comparing five immunological labeling methods using human IgG as a model antigen. Results were evaluated on the basis of the least amount of detectable antigen and background staining. The most sensitive dot-blot method was then tested for its applicability to Western blots. For both dot-blots and Western blots, the immunoalkaline phosphatase methods are more sensitive than the corresponding immunoperoxidase methods. The use of biotinylated secondary antibodies and an avidin-enzyme conjugate is recommended. Disclosure of alkaline phosphate is best achieved with naphthol AS phosphate as substrate and fast blue BB as chromogen. Peroxidase is best stained using H2O2 and diaminobenzidine (DAB). Potential endogenous enzyme activities are demonstrable by blotting methods but can be inhibited by including levamisole in the disclosure reaction medium for calf intestinal alkaline phosphatase indicators, or by incubation of blots with sodium azide and hydrogen peroxide before immunolabeling when using horseradish peroxidase indicators.

Molecular and Crystal Structure of d(CGCGmo4CG): N4−Methoxy-cytosineguanine Base Pairs in Z-DNA

1990 ◽  
Vol 9 (3) ◽  
pp. 467-469 ◽  
Author(s):  
L. Van Meervelt ◽  
M. H. Moore ◽  
P. Kong Thoo Lin ◽  
D. M. Brown ◽  
O. Kennard
Keyword(s):  
Crystal Structure ◽  
Molecular And Crystal Structure ◽  
Base Pairs ◽  
Z Dna

scholarly journals New chromogens for alkaline phosphatase histochemistry: salmon and magenta phosphate are useful for single- and double-label immunohistochemistry.

1994 ◽  
Vol 42 (4) ◽  
pp. 551-554 ◽  
Author(s):  
C Avivi ◽  
O Rosen ◽  
R S Goldstein
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Molecular Biology ◽  
Horseradish Peroxidase ◽  
Reaction Products ◽  
Immunocytochemical Detection ◽  
Hematoxylin Staining ◽  
Double Label ◽  
Biology Research

Two new substrate chromogens for alkaline phosphatase (ALP) detection have been recently synthesized for use in molecular biology research, salmon and magenta phosphate. We show here that these two chromogens have advantageous characteristics for immunocytochemistry as well. Their relatively delicate pink- and magenta-colored products do not mask the colors produced by other staining procedures. In addition, the reaction products of these substrates are insoluble in water, ethanol, and xylene, permitting the use of regressive hematoxylin staining procedures and coverslipping in permanent resin-based media. Most importantly, when these ALP substrates are used in double-label immunocytochemistry in combination with horseradish peroxidase-diaminobenzidine (HRP-DAB) and counterstained with hematoxylin, all three colors can be easily distinguished. An application using these substrates for simultaneous immunocytochemical detection of two monoclonal antibodies of different classes, in combination with hematoxylin staining, is illustrated.

scholarly journals One-step double immunolabeling of mouse interdigitating reticular cells: simultaneous application of pre-formed complexes of monoclonal rat antibody M1-8 with horseradish peroxidase-linked anti-rat immunoglobulins and of monoclonal mouse anti-Ia antibody with alkaline phosphatase-coupled anti-mouse immunoglobulins.

1991 ◽  
Vol 39 (12) ◽  
pp. 1719-1723 ◽  
Author(s):  
T Krenács ◽  
H Uda ◽  
S Tanaka
Keyword(s):  
Alkaline Phosphatase ◽  
Horseradish Peroxidase ◽  
B Lymphocytes ◽  
Enzyme Reaction ◽  
Molecular Complexes ◽  
Simultaneous Application ◽  
One Step ◽  
Reticular Cells ◽  
Secondary Antibodies ◽  
Double Immunolabeling

A novel one-step double immunolabeling method was elaborated on the basis of the simultaneous application of preformed molecular complexes of two primary antibodies with their specific secondary antibodies labeled with different enzymes. Treatment with a rat monoclonal antibody (MAb), M1-8, pre-coupled with horseradish peroxidase-linked sheep anti-rat immunoglobulins, and enzyme reaction revealed by the 3-amino-9-ethylcarbazole/hydrogen peroxide reaction, resulted in red-brown intracytoplasmic staining of interdigitating reticular cells in the lymph nodes of Balb/c mice. Another molecular complex, made of mouse anti-Ia MAb with alkaline phosphatase-linked rabbit anti-mouse immunoglobulins, applied at the same time and then developed with naphthol AS-BI-phosphate/fast blue BB as substrate, yielded blue surface staining of this cell type in addition to labeling of B-lymphocytes. The method described provides the possibility of relatively rapid double antigen detection where the binding sites of the secondary antibodies are saturated by the specific primary immunoglobulins. This approach seems to avoid nonspecific binding of primary antibodies to Fc receptors, and the unwanted binding of secondary antibodies with cell surface immunoglobulins on B-lymphocytes or with crossreactive primary antibodies used in the other sequence, if the primary antibodies and the tissue are the same or crossreactive animal species.

scholarly journals Why do A.T base pairs inhibit Z-DNA formation?

1990 ◽  
Vol 87 (12) ◽  
pp. 4630-4634 ◽  
Author(s):  
L. X. Dang ◽  
D. A. Pearlman ◽  
P. A. Kollman
Keyword(s):  
Base Pairs ◽  
Z Dna

PROTEASE NEXIN II IN HUMAN PLATELETS.

1987 ◽  
Author(s):  
W E Van Nostrand ◽  
P P Cunningham
Keyword(s):  
Growth Factor ◽  
Human Fibroblasts ◽  
Human Platelets ◽  
Polyclonal Antiserum ◽  
Single Chain ◽  
Gamma Subunit ◽  
Apparent Homogeneity ◽  
Rabbit Polyclonal Antiserum ◽  
Cultured Human Fibroblasts ◽  
Protease Nexin

Normal human fibroblasts secrete a protein into the culture medium named protease nexin II (PN II), which forms sodium do-decyl sulfate (SDS)-stable complexes with epidermal growth factor binding protein (EGF BP). These complexes then bind back to the same cells and are rapidly internalized and degraded. We recently purified PN II to apparent homogeneity and showed that it is a single chain polypeptide with an estimated molecular weight of 106 KDa. Other interesting properties of this protein include its ability to bind heparin and its stability to treatment with SDS or pH 1.5. In addition to EGF BP, PN II will also complex the gamma subunit of 7S nerve growth factor (NGF-gamma) and trypsin. Here we show that human platelets contain a protein which possesses the same properties as does PN II from cultured human fibroblasts. This platelet PN II (PL-PN II) is also a 106 KDa single chain polypeptide which can complex EGF BP, NGF-gamma and trypsin. PL-PN II also possesses the unusual stability to treatment with SDS or pH 1.5. In addition, both PN II and PL-PN II exhibit the same affinity for binding to heparin-Sepharose. Furthermore, rabbit polyclonal antiserum raised against PN II recognized PL-PN II on Western blot analysis demonstrating that both proteins are immunologically related. Treatment of fresh unactivated platelets with epinephrine or thrombin resulted in a release of PL-PN II into the supernatant suggesting that PL-PN II is stored in the platelet alpha granules.

Rab3D localizes to zymogen granules in rat pancreatic acini and other exocrine glands

1996 ◽  
Vol 271 (3) ◽  
pp. G531-G538 ◽  
Author(s):  
H. Ohnishi ◽  
S. A. Ernst ◽  
N. Wys ◽  
M. McNiven ◽  
J. A. Williams
Keyword(s):  
Polyclonal Antiserum ◽  
Secretory Cells ◽  
Apical Region ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Base Pairs ◽  
Pancreatic Acini ◽  
Pcr Product ◽  
The Family ◽  
Granule Membrane

Rab3 proteins are members of the family of Ras-like monomeric GTP-binding proteins that have been implicated in secretion in neuronal cells. Although an isoform of Rab3 has been assumed to exist in pancreatic acini, its identity has not yet been established. We now report that Rab3D is present in rat pancreatic acini and is localized to the zymogen granule membrane. Reverse transcription-polymerase chain reaction (PCR) was used with primers based on mouse Rab3D to amplify Rab3D from rat pancreas. The PCR product without primer sites consisted of 580 base pairs and was 94% identical to the mouse Rab3D cDNA sequence previously cloned from adipocytes. Western blotting with a polyclonal antiserum raised against Rab3D-specific carboxyterminal amino acids identified Rab3D in rat pancreatic acini and revealed its concentration on zymogen granule membranes. Immunocytochemistry of pancreatic lobules showed that Rab3D localized to the apical region in a pattern similar to amylase. Confocal fluorescence microscopy of lobules double immunolabeled with antibodies to Rab3D and the granule membrane marker protein glycoprotein-2 (GP-2) revealed a similar localization of these proteins to zymogen granules. Immunocytochemistry also revealed the presence of Rab3D in chief and enterochromaffin-like cells in the stomach, acinar cells in lacrimal and parotid gland, and Paneth cells in the intestine. These results show that Rab3D is expressed in rat pancreatic acini and other exocrine secretory cells. Its location implies it may be involved in regulated exocytosis.

Comparison of alkaline phosphatase and horseradish peroxidase conjugated antisera in the ELISA test for antibodies to reovirus 3, mouse hepatitis and Sendai viruses

1981 ◽  
Vol 15 (1) ◽  
pp. 69-74 ◽  
Author(s):  
P. Carthew ◽  
J. Gannon ◽  
I. Whisson
Keyword(s):  
Alkaline Phosphatase ◽  
Horseradish Peroxidase ◽  
Virus Type ◽  
Sendai Virus ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Elisa Test ◽  
Serological Response

The method of enzyme-linked immunoadsorbent assay (ELISA) has been applied to the detection of antibodies to reovirus 3, Sendai virus and mouse hepatitis virus (type 1), and the serological response of mice after infection has been followed for 28 days to investigate the earliest appearance of ELISA titres. This has been compared to the appearance of haemagglutination-inhibiting and complement-fixing antibodies. Alkaline phosphatase conjugated antiserum produces the most sensitive and convenient ELISA for the murine viruses examined.

scholarly journals Identification of cell surface receptors for the Act-2 cytokine.

1990 ◽  
Vol 172 (1) ◽  
pp. 285-289 ◽  
Author(s):  
M Napolitano ◽  
K B Seamon ◽  
W J Leonard
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
K562 Cells ◽  
Peripheral Blood Lymphocytes ◽  
Polyclonal Antiserum ◽  
Cell Surface Receptors ◽  
Surface Receptors ◽  
Rabbit Polyclonal Antiserum ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd

We have identified cell surface receptors for Act-2, a secreted protein expressed upon activation of T cells, B cells, and monocytes. Although 125I-Act-2 showed little, if any, specific binding to resting peripheral blood lymphocytes (PBL) receptors were readily detected on PHA/PMA-activated PBL and a variety of cell lines including MT-2, HL60, DMSO differentiated HL60, HeLa, and K562 cells. The equilibrium dissociation constant (Kd) is 3-12 nM for MT-2, K562, and PBL activated with PHA/PMA for 40-80 h. We have also identified a rabbit polyclonal antiserum that can block Act-2 binding to its receptors. The ability to detect specific Act-2 receptors and the development of a blocking antiserum should prove valuable in efforts to molecularly clone the Act-2 receptor and to dissect the biological actions of Act-2.

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