Indirect Labeling Method Research Articles

Protein Arrays: Sandwich Approach for Analyzing Complex Solutions

INTRODUCTIONProtein arrays can be used to quantitate the abundance and modification states of proteins in complex mixtures. For this application, it is necessary to array-capture reagents, such as antibodies, on the slides and then use these molecules to specifically capture their cognate antigens from solution. The investigator has a choice in either labeling the proteins that are under study or detecting the captured proteins by an indirect labeling method. In the latter case, which is detailed in this protocol, each protein is captured by one antibody (or other capture reagent) and then detected in a second step with a second reagent. The second reagent, which in this case is also an antibody, recognizes the protein at a site that does not overlap with the recognition site of the capture reagent. In this sandwich approach, the second reagent is labeled and so provides the signal. The advantage of this approach is that it does not require labeling the proteins themselves. Moreover, additional specificity is gained by using two different reagents for each protein. The disadvantage, however, is that it is more difficult to assemble matched pairs of antibodies for each protein of interest.

CDNA Labeling Strategies for Microarrays Using Fluorescent Dyes

AbstractMicroarray analysis has become a key experimental tool in the study of genome‐wide patterns of gene expression. The labeling step of target molecules such as cDNA or cRNA plays a key role in a microarray experiment because the amount of mRNA is measured indirectly by the labeled molecules. In this paper, the most widely used cDNA labeling strategies in microarray experiments are reviewed in detail, including direct labeling and indirect labeling methods along with a discussion of the merits and disadvantages of these methods. Furthermore, various RNA amplification approaches were surveyed to obtain a target nucleic acid sufficient for microarray experiments from minute amounts of mRNA. Finally, the labeling strategies of commonly used microarray platforms (e.g., Affymetrix GeneChip®, CodeLink™ Bioarray, Agilent and spotted microarrays) were compared.

Preparation of 18F labeled amino acid O-(2-[18F]fluoroethyl)-L-tyrosine using indirect and direct labeling methods

18 F-labeled amino acid O-(2 -[ 18 F]fluoroethyl) -L-tyrosine ([ 18 F]FET) has been used as an in vivo positron emission tomography (PET) tracer for tumor metabolitic imaging. Here, t he synthesis of [ 18 F]FET using two different methods is reported. The first Method (I) for the [ 18 F]FET synthesis was an indirect labeling route , which was a two -step reaction consisting of fluorination of 1,2 -bis(tosyloxy)ethane and fluoroalkylation of un protected L -tyrosine. The second Method (II) was a direct labeling route which was the direct nucleophilic radiofluorination of the protected precursor N-BOC -(O-(2 -tosyloxyethyl)) -L-tyrosine methyl ester, followed by a rapid removal of the protecting group . For the first method, the radiochemical yield was about 45% at the end of synthesis (EOS), and the radiochemical purity was over 97%. The radiochemical yield in the second method was 40% (EOS) on an average, and the radiochemical purity was over 96%. Mic rowave heating was also introduced into the synthesis of [ 18 F]FET. For Method I, each of two steps could be completed within 2 –3 minutes under microwave conditions and the radiochemical yields were 85% and 95%, respectively, which was reasonably high and r eproducible. For Method II, the highest labeling efficiency in the radiofluorinated step was 37% in the synthesis time of 3 minutes under microwave conditions.

Clinical utility of assessing ZAP‐70 and CD38 in chronic lymphocytic leukemia

Clinical utility of assessing ZAP‐70 and CD38 in chronic lymphocytic leukemia

Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4α

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates many genes that are preferentially expressed in liver. Mice lacking hepatic expression of HNF4alpha (HNF4alphaDeltaL) exhibited markedly increased levels of serum bile acids (BAs) compared with HNF4alpha-floxed (HNF4alphaF/F) mice. The expression of genes involved in the hydroxylation and side chain beta-oxidation of cholesterol, including oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase (CYP8B1), and sterol carrier protein x, was markedly decreased in HNF4alphaDeltaL mice. Cholesterol 7alpha-hydroxylase mRNA and protein were diminished only during the dark cycle in HNF4alphaDeltaL mice, whereas expression in the light cycle was not different between HNF4alphaDeltaL and HNF4alphaF/F mice. Because CYP8B1 expression was reduced in HNF4alphaDeltaL mice, it was studied in more detail. In agreement with the mRNA levels, CYP8B1 enzyme activity was absent in HNF4alphaDeltaL mice. An HNF4alpha binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4alpha-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4alpha plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain beta-oxidation of cholesterol in vivo.

Use of Synaptophysin Immunohistochemistry in Intestinal Motility Disorders

The purpose of this study was to describe synaptophysin (SY) immunoreactivity in colonic specimens from patients with Hirschsprung's disease (HD), chronic constipation (CC), or anal atresia (AA). This membrane protein is specific for the synaptic vesicles in the central and peripheral nervous system and responsible for neurotransmission. Biopsy specimens of the intestinal wall were obtained from 18 patients (age range, 2 days to 7 years). Immunohistochemistry was performed using rabbit anti-human antibodies specific for synaptophysin (DAKO). In the ganglionic colon of HD patients and others the immunoreactivity of SY-positive synapses was abundantly present in the smooth muscle layers. Distinct immunoreactivity showed ganglion cells and nerve fibers inside circular and longitudinal muscle layers. In some non-HD patients' colonic specimens SY-positive synapses were present in the muscularis mucosae. In the aganglionic colonic segment of HD-patients no immunoreactivity of synapses and ganglions was seen. In the transition zone, where ganglion cells appeared sporadically, synapses were very rarely present. In two patients from the CC group the amount of visualized synapses was clearly smaller and the concentration of ganglion cells within ganglions in these cases was much lower than usual (but still within normal ranges). In the AA group in the distal part of the atretic rectum (at the place where the fistula was cut) SY-positive synapses were present in smooth muscle layers and small dysplastic ganglions were seen in the submucosal and muscular region, but not in large numbers. These patients had a normal distribution of ganglion cells and synapses at the place of colostomy. Synaptophysin immunohistochemistry is an indirect labeling method with a high detection rate for intestinal ganglion cells by demonstrating their synapses. Changed intestinal distributions of SY-positive synaptic vesicles usually accompany colonic ganglion cell disorders. The pattern of SY-positive synapses distribution in circular and longitudinal colonic muscles and intermuscular ganglions can reflect functional disturbances of large bowel motility and could be helpful in the description of the innervation status of colonic specimens in HD patients.

Detection of Single Bacterial Pathogens with Semiconductor Quantum Dots

Semiconductor quantum dots (QDs) have been used in a simple fluorometric assay to detect single cells of the pathogenic Escherichia coli O157:H7 serotype. Composed of CdSe/ZnS core/shell QDs conjugated to streptavidin, this system exhibits 2 orders of magnitude more sensitivity than a similar assay using a common organic dye. Selectivity for this pathogenic bacterial strain over a common lab strain (E. coli DH5alpha), which is gained from the use of specific biotinylated antibodies, is also demonstrated for QD labeling. Under continuous excitation, these QDs retain high fluorescence intensities for hours, whereas a typical organic dye bleaches within seconds, allowing for more rapid and accurate identification of E. coli O157:H7 in single-cell fluorescence-based assays. This indirect QD labeling method, based on antibody-antigen and streptavidin-biotin interactions, is flexible enough to expand to other systems and has great potential for use in simultaneous multicolor detection schemes.

Evaluation of the Performance of Two Carbodiimide-Based Cyanine Dyes for Detecting Changes in mRNA Expression with DNA Microarrays

Microarrays have been extensively used to investigate genome-wide expression patterns. Although this technology has been tremendously successful, several practical issues would benefit from improvements in design. Here we describe a novel, efficient labeling methodology that uses carbodiimide-linked cyanine dyes to directly chemically label cDNA derived from mouse total RNA. Using this protocol, it takes only 10 min at 70 degrees C to complete the cDNA labeling reaction. The directly labeled cDNAs can then be hybridized to 70-mer mouse oligonucleotide arrays for expression profiling studies. Microarray analyses indicate that these cDNAs are uniformly labeled and produce higher signal intensities than conventional enzymatic direct labeling methods and comparable signal intensities to those obtained by conventional indirect labeling methods. Furthermore, verification of our microarray data using a reverse transcription PCR (RT-PCR) method indicates good agreement between the two methods. Thus, we conclude that our simplified cyanine-carbodiimide labeling method, which does not rely on the incorporation of modified nucleotides, will provide a reliable, quicker, and potentially cheaper alternative to established labeling techniques for gene expression analyses.

Evaluation of Transcript Labeling Techniques and Development of a Membrane-based Parallel Gene Expression Assay

An inexpensive alternative to micro arrays was developed to examine the expression of small sets of genes in parallel and used to compare direct and indirect transcript labeling methods. Psoralen-biotin direct labeling was ~10-fold less sensitive than reverse transcriptase-biotin-dUTP labeling, but the enzymatic method generated higher levels of background noise in miniarray hybridizations. The reproducibility of hybridization intensities was the same for the two labeling methods, although differences in signal intensities for several genes were observed. The miniarray hybridization assay was validated using the known responses of mycobacteria to heat shock, exposure to isoniazid and growth phase. Expression profiles were generated for 14 Mycobacterium smegmatis and 26 M. tuberculosis genes. The transcriptional response to isoniazid and peculiar regulation of acr in different growth phases were confirmed and a potential role of oxidative stress enzymes in the heat shock response was revealed. The miniarray system was also used to demonstrate that an RNA stabilizing reagent, RNALaterTM , was effective in inhibiting both RNA degradation and transcription in mycobacteria, which may prove useful when significant manipulation of the bacteria are required prior to RNA extraction such as in experimental infections of cell cultures or animals.

Changes in the expression of angiotensin II type 1 receptor in the development of liver fibrosis

To detect the expression of angiotensin II type 1 receptor (AT1R) in the different stages of human liver fibrosis. The morphology and ultrastructure of hepatic stellate cells (HSC) in hepatic sinusoids were studied by transmission electron microscopy, collagen I (col I) was tested by immunohistochemical method, an indirect immunofluorescence labeling method was used to detect AT1R, and semiquantitative RT-PCR was used to detect AT1R mRNA. Positive expression of AT1R was mainly distributed in the periphery of hepatic lobules and in the cytoplasm of HSC in the sinusoids. In normal liver tissue from 12 cases, positive expression of AT1R was seen in 8, whereas in 18 fibrotic livers, all showed positive expression. Cells that positively expressed AT1R were significantly more abundant in the fibrotic liver group than in the normal liver group (P < 0.001), and were significantly increased with an increase in the collagenous surface area. The relative expression level of AT1R mRNA in the fibrotic liver group was also higher than in the normal liver group (P < 0.01). The expressions of both AT1R and AT1R mRNA increased significantly with the degree of liver fibrosis, so angiotensin II and its receptor are probably important in the development of liver fibrosis.

Correlation between the expression of Fas, Bcl-2 in peripheral blood lymphocytes and the level of IFN-gamma, IL-4 in serum of patients with condyloma acuminata.

In order to investigate the correlation between the expression of the apoptotic regulatory proteins (Fas, Bcl-2) in peripheral blood lymphocytes (PBLC) and the level of IFN-gamma and IL-4 in serum of the patients with condyloma acuminata (CA) in the immune pathogenesis of CA, indirect immunofluorescence labeling method of flow cytometer and solid sandwich ELISA method were performed for detecting the expression of Fas, Bcl-2 in PBLC and the level of IFN-gamma and IL-4 in serum of 60 cases of CA. The results showed the expression level of Fas in PBLC of CA was significantly higher than in the normal control group, but the expression level of Bcl-2 was significantly lower (both P<0.01). The level of IFN-gamma in serum of CA was significantly lower than in the normal control group (P<0.01), but IL-4 was significantly lower (both P<0.01). The expression of Fas in PBLC had a negative correlation with the level of IFN-gamma in serum of patients with CA, but had a positive correlation with the level of IL-4; The expression of Bcl-2 had a positive correlation with the level of IFN-gamma, but had a negative correlation with the level of IL-4. All the correlation coefficients had significant difference by t test (P<0.01). It was suggested abnormal apoptosis in PBLC, the suppressed secretion of the TH1-associated cytokines (eg: IFN-gamma) and the increased secretion of the TH2-associated cytokines (eg: IL-4) existed in the patients with CA and might play an important role in the immune pathogenesis of CA.

145 Preliminary Results of Cytoskeletal Components in Various Red Algae Using Confocal Laser Scanning Microscopy

Data concerning the cytoskeletal components of red algae are scant. The goal of this ongoing comparative survey is to develop a more complete characterization of the red algal cytoskeleton, and subsequent elucidation of its function, using representative taxa belonging to major evolutionary lineages within the Rhodophyta. Preliminary data were obtained using enzyme digestion of cell walls and detergent rinses with direct (phalloidin) and indirect (monoclonal antibodies) labeling methods for microfilaments and tubulin, respectively. Samples were viewed using confocal laser scanning microscopy. Results will be discussed in light of the more thoroughly understood cytoskeletal system reported for green algae.

Identification of multiple neuropeptide Y receptor subtypes in the human frontal cortex

Recently, we found abundant mRNA and binding sites for neuropeptide Y Y 1-like receptors in the human cerebral cortex. However, an earlier study using indirect labeling methods failed to detect substantial neuropeptide Y 1-like receptor binding in numerous areas of the human brain, including the cerebral cortex. To resolve the disparity in these findings, we characterized the neuropeptide Y receptor subtypes labeled with [ 125 I ]peptide YY in homogenates of human frontal cortex. Competition experiments using 100 pM [ 125 I ]peptide YY binding to human frontal cortex homogenates indicated predominantly neuropeptide Y Y 2 receptors are labeled with this concentration of ligand. However, saturation analysis of [ 125 I ]peptide YY binding to frontal cortex membranes resulted in isotherms best characterized by a two-site fit. Binding of [ 125 I ]peptide YY to the high affinity ( K d=40 pM) binding site was prevented using a 100 nM concentration of the neuropeptide Y Y 2 receptor agonist peptide YY-(3–36). By masking the higher affinity site, we found a low affinity [ 125 I ]peptide YY binding site ( K d=1.4 nM) exhibiting a pharmacology consistent with a neuropeptide Y Y 1-like receptor. It appears that neuropeptide Y Y 2 receptors are the predominant subtype labeled with low concentrations of [ 125 I ]peptide YY and that the neuropeptide Y Y 1 receptor is a low affinity [ 125 I ]peptide YY binding site in the human frontal cortex.

Analysis of NPY Receptor Subtypes in the Human Frontal Cortex Reveals Abundant Y 1 mRNA and Binding Sites

Statnick, M. A., D. A. Schober, N. G. Mayne, J. P. Burnett and D. R. Gehlert. Analysis of NPY receptor subtypes in the human frontal cortex reveals abundant Y 1 mRNA and binding sites. Peptides 18(1) 137–143, 1997.—Receptors for neuropeptide Y (NPY) are widely distributed throughout the mammalian brain. Using indirect labeling methods, the human brain was reported to contain predominantly the Y 2 receptor subtype, whereas the rat brain contains a mixture of Y 1 and Y 2 receptor subtypes. To more accurately assess NPY receptors in the human brain, we used the Y 1- and Y 2-selective radioligands [ 125I][Leu 31,Pro 34]PYY and [ 125I]PYY(3–36), respectively, to examine NPY receptors in the human frontal cortex. Contrary to an earlier report, abundant Y 1 binding sites were found in homogenates of human frontal cortex. Moreover, saturation analysis showed similar densities of both Y 1 ( K d = 433 ± 36 p M, Bmax = 313 ± 15 fmol/mg protein) and Y 2 ( K d = 444 ± 39 p M, Bmax = 458 ± 22 fmol/mg protein) receptor subtypes in the human frontal cortex. Subsequently, Northern blot analysis revealed abundant expression of Y 1 mRNA, with very low levels of Y 2 mRNA, in cerebral cortex and in other areas of the human brain. These findings were confirmed by competitive RT-PCR in the human frontal cortex. Therefore, it appears that Y 1 binding sites and mRNA are expressed abundantly in the human frontal cortex and, unlike earlier findings, suggest that the human brain contains a mixture of Y 1 and Y 2 receptor subtypes.

5292636 Therapeutic and diagnostic methods using soluble T cell surface molecules

Differences in expression of cytochrome P450 forms and their functions in different organs and cell types could determine the response of those cells and organs to xenobiotics. Recently, we described the cellular localization of cytochrome P450IA1 (P450E) induced in 10 organs or organ systems of the fish, Stenotomus chrysops (scup) treated with 3,3′,4,4′-tetrachlorobiphenyl or with 2,3,7,8-tetrachlorodibenzofuran. (R. M. Smolowitz, M. E. Hahn, and J. J. Stegeman, Drug Metab. Dispos. 19, 113, 1991). Here we describe the presence and localization of P450IA1 in organs of scup sampled directly from an environment contaminated by chlorinated biphenyls and dibenzofurans, the outer New Bedford Harbor of Massachusetts. Western blot analysis of microsomes from selected organs (liver, kidney, gill, and heart), using monoclonal antibody 1-12-3, revealed induced levels of P450IA1 in each. The localization of P450IA1 in these and other organs was determined in sections prepared by standard histological methods and stained with MAb 1-12-3 in an indirect peroxidase labeling method. P450IA1 was detected in multiple cell types in liver, including hepatic, pancreatic, and vascular tissue. Kidney and gut also showed prominent P450IA1 levels in epithelial structures and in vascular endothelial cells. Specific staining was detected in endothelial cells, but not other cell types, in heart, gill, spleen, testis, ovary, nose, and brain. In heart, the staining was present in the endocardium of atrium and ventricle, and endothelium of the coronary vasculature and great vessels. The results demonstrate that P450IA proteins are induced in many organs of fish exposed to environmental chemicals in the wild, with patterns of cellular localization like those seen in fish experimentally treated with known inducers. The strong staining of P450IA1 in endothelial cells in all organs examined supports experimental results indicating that endothelium is a major site of P450IA1 induction. Our results indicate further that immunohistochemistry is a useful method for detecting P450 induction as a biomarker for exposure.

Immunohistochemical localization of environmentally induced cytochrome P450IA1 in multiple organs of the marine teleost Stenotomus chrysops (scup)

Differences in expression of cytochrome P450 forms and their functions in different organs and cell types could determine the response of those cells and organs to xenobiotics. Recently, we described the cellular localization of cytochrome P450IA1 (P450E) induced in 10 organs or organ systems of the fish, Stenotomus chrysops (scup) treated with 3,3′,4,4′-tetrachlorobiphenyl or with 2,3,7,8-tetrachlorodibenzofuran. (R. M. Smolowitz, M. E. Hahn, and J. J. Stegeman, Drug Metab. Dispos. 19, 113, 1991). Here we describe the presence and localization of P450IA1 in organs of scup sampled directly from an environment contaminated by chlorinated biphenyls and dibenzofurans, the outer New Bedford Harbor of Massachusetts. Western blot analysis of microsomes from selected organs (liver, kidney, gill, and heart), using monoclonal antibody 1-12-3, revealed induced levels of P450IA1 in each. The localization of P450IA1 in these and other organs was determined in sections prepared by standard histological methods and stained with MAb 1-12-3 in an indirect peroxidase labeling method. P450IA1 was detected in multiple cell types in liver, including hepatic, pancreatic, and vascular tissue. Kidney and gut also showed prominent P450IA1 levels in epithelial structures and in vascular endothelial cells. Specific staining was detected in endothelial cells, but not other cell types, in heart, gill, spleen, testis, ovary, nose, and brain. In heart, the staining was present in the endocardium of atrium and ventricle, and endothelium of the coronary vasculature and great vessels. The results demonstrate that P450IA proteins are induced in many organs of fish exposed to environmental chemicals in the wild, with patterns of cellular localization like those seen in fish experimentally treated with known inducers. The strong staining of P450IA1 in endothelial cells in all organs examined supports experimental results indicating that endothelium is a major site of P450IA1 induction. Our results indicate further that immunohistochemistry is a useful method for detecting P450 induction as a biomarker for exposure.

Involvement of nucleus in the maturation of dengue-2 virus in C6/36 cells

The involvement of nucleus in the maturation processes of Dengue-2 virus in a mosquito cell line, C6/36 cells, has been identified by the electron microscopy and immunocytochemistry. The C6/36 cells were obtained from ATCC and maintained in MEM culture medium containing 10% fetal bovine serum at 28°C. The cell suspensions or cells grown on teflon-coated coverslips were infected with Dengue-2 virus (107/ml) for various time periods of 2 hours, 3, 6, 8, and 10 days. The cells were then fixed in buffered 1.5% glutaraldehyde, and washed in acetone before immunolabeled with monoclonal antibody. An indirect immunocytochemical labeling method of avidin-biotin complex (ABC) conjugated with peroxidase or gold particles (20 nm in diameter) and a flat embedding technique were used to localize the virus particles.At early stages of infections (before 3 days), there were no virion particles detected. After 6 days and on of infections, cytopathic effect (CPE) was observed and showed positive immuno-peroxidase reactions under the light and electron microscopies.

In vivo assembly of chromatin on pBR322 sequences cloned into yeast plasmids

In order to study the in vivo assembly of chromatin on prokaryotic DNA templates, we have transformed yeast cells with plasmids pAJ50 and pRB58, which contain pBR322 sequences. In both cases nucleosomes are assembled in vivo on pBR322 DNA, although the nucleosomes are not homogeneous in size. To explore whether there is any preference for nucleosome assembly along pBR322 sequences, we have used an indirect end labeling method. The results indicate that most nucleosomes are placed at random on pBR322, although the probability for histone octamers to interact with some short regions is somewhat reduced. These regions coincide with sequences in which the frequency distribution of nucleosomes reconstituted in vitro (E. Caffarelli et al. (1988) Eur. J. Biochem. 171, 497-501) is low. Sequence determinants that direct chromatin assembly in vitro seem thereby to act to some extent in vivo.

Schistosoma mansoni: Origin and expression of a tegumental surface antigen on the miracidium and primary sporocyst

A monoclonal antibody, recognizing a carbohydrate epitope associated with several tegumental surface components on Schistosoma mansoni primary sporocysts, was used to follow tegumental formation during transformation of the miracidium to sporocyst and its subsequent development in vitro and in vivo. Indirect fluorescent antibody and direct immunogold labeling methods confirm a structural connection between the intercellular ridges and a submuscular, multinucleate syncytium in the miracidium. Immunoreactive vesicles within this latter system directly contribute to elaboration of the tegumental surface membrane, through the process of membrane fusion. Lateral expansion of intercellular ridges by vesicular fusion ultimately result in fully transformed sporocysts exhibiting vesicular membrane epitopes as prominent tegumental surface components. Light microscopical and ultrastructural observations, together with Western immunoblot analyses, suggest a gradual depletion of intracellular and surface immunoreactive material of vesicular origin in primary sporocysts grown in culture for up to 12 days. In contrast, similar immunoreactive vesicles appear to be continuously synthesized throughout in vivo primary sporocyst development. Monoclonal antibody reactive epitopes appear to be uniquely expressed in the miracidium/primary sporocyst since similar molecules are absent from daughter sporocysts, cercariae, adults, and snail tissues.

Post-separation detection of nucleic acids and proteins by neutron activation.

We describe approaches to neutron activation analysis and their application to post-separation autoradiographic detection of biological compounds. Specifically, we have extended the use of a "direct-labeling" method to the post-separation detection of DNA after gel electrophoresis and to the detection of nucleotides separated by TLC. In addition, we describe a more generally applicable "indirect-labeling" method in which separated compounds of interest are selectively bound to ligands containing highly neutron-activatable elements, such as manganese (55Mn), europium (151Eu), or dysprosium (164Dy), and then irradiated with thermal neutrons. This method is illustrated with nucleotides separated by TLC and with proteins separated by polyacrylamide gel electrophoresis. In contrast to the direct-labeling approach, the indirect-labeling method can be adapted to detect any class of substances for which a highly neutron-activatable, selectively binding ligand is available. The theoretically achievable sensitivity of the indirect-labeling method is in the attomole (10(-18) mol) range.