Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.     

Detection of hepatitis B virus DNA in human serum samples: use of digoxigenin-labeled oligonucleotides as modified primers for the polymerase chain reaction.

Nonradioactive techniques have been used for the direct detection of hepatitis B virus DNA in human serum samples. A comparison of two different systems using digoxigenin-labeled DNA probes is presented. Furthermore, oligonucleotides containing one molecule of the hapten digoxigenin at the 5'-end were prepared and used as primers for the polymerase chain reaction. Amplified DNA can be directly analyzed with anti-digoxigenin Fab fragments labeled with alkaline phosphatase and chemiluminescent substrates.     

New,sensitive, radioactive-free bioluminescence-enhanced detection system in protein blotting and nucleic acid hybridization

A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blotting and nucleic acid hybridization is described. The method utilizes antibodies conjugated with alkaline phosphatase or nucleotide probes complexed with alkaline phosphatase. Then the alkaline phosphatase takes part in a reaction by releasing D -luciferin (Photinus pyralis) from D -luciferin-O-phosphate. Liberated D -luciferin reacts with luciferase, ATP and oxygen under light emission. Light is measured using the Argus-100 a photon counting camera system or photographic films. Bound alkaline phosphatase conjugated antibodies or hybridized nucleotide probes can be visualized. The limit of detection is at present 5 to 50 fg of protein (IgG), corresponding, for example to 30 to 300 × 10^?21 mol. This means a much higher sensitivity of the detection system in comparison to systems used at present. Experiments concerning nucleic acid hybridization and visualization of the emitted light by a photon counting camera (Argus-100) are under investigation.     

Histochemical study of a number of hydrolases,including a new acid phosphatase,in various tissues of men and mice

Summary Two acid phosphatases have been demonstrated histochemically in mouse ventral prostate, seminal vesicles, coagulating glands, and liver and in human prostate. The first is the lysosomal acid phosphatase demonstrable by the Gomori technique. The second differs from thisβ-glycerophosphatase in that it splits naphthol AS phosphates but notβ-glycerophosphate; it has a different histochemical pH optimum and it is not inhibited by MoO₄ or NaF. The enzyme does not represent the “tail” of alkaline phosphatase activity as it is not inhibited by inhibitors of alkaline phosphatase and it has a different localization in liver and in human prostate. The enzyme may be membrane-bound but a lysosomal localization has still to be confirmed.     

Combined immunohistochemical staining for surface IgD and T-lymphocyte subsets with monoclonal antibodies in human tonsils

The aim of the present paper is to detect two different antigens simultaneously in a single slide. In cryostat sections of human tonsils, B-lymphocytes of follicle mantle-bearing surface IgD were immunostained with the alkaline phosphatase method using monoclonal anti IgD. The subsequent staining for T-lymphocyte subsets (T-helper and T-suppressor lymphocytes) was performed again with the alkaline phosphatase method using one of the monoclonal antibodies OKT 4, OKT 8, Leu 3a, Leu 2a. The best results with the alkaline phosphatase method were achieved using naphthol AS phosphate and Fast Blue BB for the revelation of the first antigen and naphthol AS-BI phosphate and diazotized New Fuchsin for the second.     

Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system)

A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.     

Alkaline phosphatase inhibitors as labels of DNA probes

A new approach to nucleic acid labeling was developed by preparing bifunctional reagents containing, in addition to the DNA-linking group, a competitive inhibitor of the chromogenic enzyme alkaline phosphatase. The nucleic acids labeled in such a way were able to bind themselves to the enzyme, whose activity was restored in the presence of a chromogenic substrate. Five phosphonic-acid-containing reagents were synthesized and coupled to linearized pBR322 plasmid DNA by different condensation methods. Eight probes thus obtained were assayed in a modified dot-blot detection procedure obtaining the best nucleic acid detection sensitivity of 25 pg. Finally, five of the above probes were tested in hybridization experiments, reaching sensitivity of 50 pg.     

基于纳米金复合探针的基因芯片膜转印检测法

建立了一种基于纳米金复合探针的基因芯片膜转印核酸检测新方法。首先,用纳米金颗粒同时标记检测探针P2和两种长短不同且生物素化的信号探针 (T10,T40),其中检测探针与靶DNA 5￠端互补,两种信号探针起信号放大作用。当靶DNA分子存在时,芯片表面捕捉探针P1 (与靶DNA分子3￠端互补) 通过碱基互补配对原则结合靶DNA分子,将其固定于芯片上,同时检测探针通过与靶DNA 5￠端互补配对将纳米金复合探针结合于芯片表面,结果在芯片表面形成“三明治”结构,后通过链霉亲和素-生物素反应,使芯片表面对应有靶DNA分子的部位结合上碱性磷酸酶,最后利用BCIP/NBT显色系统使芯片表面信号结果镜面转印至尼龙膜表面。当检测探针和信号探针摩尔比为1∶10,T10和T40摩尔比为9:1时可以检测1 pmol/L合成靶DNA分子或0.23 pmol/L结核分枝杆菌16S rDNA PCR扩增产物,检测结果通过普通的光学扫描仪读取或肉眼直接判读信号有无。本芯片检测系统灵敏度高,操作方法简单、快速,不需要特殊仪器设备,在生物分子的检测方面具有较高的应用价值。     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

A simplified lysis method allowing the use of biotinylated probes in colony hybridization

A method is described for the lysis of bacterial cells grown on nitrocellulose filters which allows the use of nonradioactive (biotin labeled) probes in colony hybridization. Used in conjunction with a colorimetric assay involving streptavidin and alkaline phosphatase this lysis method allows the detection of clones containing a target nucleic acid sequence. Sites of positive hybridization produce dark blue-purple signals, while nonreacting clones give very light blue signals. The occurrence of false-positive and background signals is minimal. With pBR322 as the target sequence it was possible to detect approximately 20 clones in the presence of 10(5) plasmid-free cells. It was also possible to detect low-frequency plasmid-free cells in a population of clones containing the target sequence.     

Non-radioactive detection of nerve growth factor receptor (NGFR) mRNA in rat brain using in situ hybridization histochemistry.

Radioactively labeled RNA probes in conjunction with in situ hybridization histochemistry have become a useful method for studying gene expression in the central nervous system. We used digoxigenin-labeled uridine triphosphate to synthesize cRNA probes for localization of nerve growth factor receptor (NGFR) mRNA in the rat basal forebrain. Detection of cells containing digoxigenin-labeled NGFR mRNA was accomplished using a digoxigenin antibody conjugated with alkaline phosphatase. NGFR mRNA-positive cells were distributed in three major cell groups in the basal forebrain: the medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. This technique provides a rapid and sensitive method for high-resolution detection of mRNA species in the central nervous system, as well as the potential for co-localization of two different mRNA species within individual cells.     

Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy

Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections This revised version was published online in November 2006 with corrections to the Cover Date.     

Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.

A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.     

Acid phosphatase and esterase activity in orchid mycorrhiza

Summary A cytochemical study was made to examine the possibility that acid phosphatase may be specifically involved in the digestion of endophytic hyphae in orchid mycorrhiza. Esterase activity was studied for comparison. Frozen sections of unfixed or glutaraldehyde-fixed protocorms of Dactylorhiza purpurella infected by Thanatephorus cucumeris (Rhizoctonia solani) were reacted for acid naphthol AS BI phosphatase, acid -glycerophosphatase or naphthol AS D esterase.A marked increase in particulate acid naphthol AS BI phosphatase activity was observed during infection of host, central, parenchyma cells shortly before hyphae lysed; a diffuse reaction of high activity was localised on lysed fungus. Acid -glycerophosphatase was present at particulate sites only in fungal cytoplasm and as a diffuse reaction on lysed fungus.Naphthol AS D esterase showed highest activity at hyphal apices. Esterase seems to be associated with growth and differentiation of hyphae in orchid cells, rather than lysis of the fungus.     

Cytofluorometric quantification of the activity and reaction kinetics of acid phosphatase

Synopsis This paper describes how fluorogenic substrates derived from naphthol AS can be used for the microscopic demonstration and cytofluorometric quantification of the activity and reaction kinetics of acid phosphatase in single living cells.A special study has been made of acid naphthol AS-BI phosphatase. However, the method can be extended to other hydrolytic enzymes. The method is sensitive and accurate because: quantification of very low enzyme activity is possible; the reaction kinetics can be evaluated with a good degree of precision inasmuch as the initial reaction velocity is derived over short times; there is an absence of distributional error; and the errors due to extra-cellular diffusion of the hydrolysed substrate, to photo-decomposition, and to autofluorescence can be contained within very narrow limits.The procedures for determining enzyme activity and reaction kinetics, and the instrumental characteristics and devices required for carrying out these measurements, are described. Some possible applications are indicated.     

A study of acid phosphatase in mouse kidney using naphthol AS/BI phosphate as a substrate

Summary A kinetic study of mouse kidney acid phosphatase has been performed using an application of the histochemical method ofBurstone (1958a, b). The suitability of the use of naphthol AS/BI phosphate as a substrate for biochemical assays of acid phosphatase has been ascertained. However, the rate of inhibition of the enzyme by sodium molybdate and sodium fluoride suggests that naphthol AS/BI phosphate may represent a substrate for an acid phosphatase different from-glycerophosphatase.     

Nonradioactive labeling and high-sensitive detection of PCR products

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactivc labeled nucleotides during PCR byTaq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin-(DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin-labeled amplification products as hybridization probes, sensilivities in the 0.1-pg range are obtained in Southern blot procedures.