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PubMed2002

[Establishment and evaluation of the method for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase].

OBJECTIVE: To establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe). METHODS: The probe that purified HBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP was used in the hybridization assay. HBV DNA was detected by autoradiography on the film. The test compared the chemiluminescen dot blot hybridization assay for 80 samples with digoxigenin-labeled HBV DNA probe detective method. The correlation of 70 samples test results between fluorescent quantitative HBV DNA PCR method and dot blot hybridization assay by AlkPhos Direc probe was analysed. RESULTS: The sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least. The coincidence was 100% compared with digoxigenin-labeled HBV DNA probe detection. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR (QPCR) method and dot blot hybridization assay by AlkPhos Direc probe was 0.98 (P<0.01). CONCLUSIONS: The method detecting HBV DNA in serum by HBV DNA AlkPhos Direc probe is sensitive and specific. The results between two methods with AlkPhos Direc and digoxigenin-labeled HBV DNA probe are coincident completely. The correlation of HBV DNA quantitative results between fluorescent QPCR method and dot blot hybridization assay by AlkPhos Direc probe is satisfactory.

Alkaline Phosphatase↗
PubMed2003

Assessment of a method for detecting serum HBV DNA with HBV DNA probe labelled directly by alkaline phosphatase.

OBJECTIVE: To assess a sensitive and specific technique for detecting serum HBV DNA with an HBV DNA probe labelled directly by alkaline phosphatase (AlkPhos Direc probe). METHODS: AlkPhos Direc probe was prepared with purified HBV DNA labelled directly by alkaline phosphatase. The probe and chemiluminescent substrate CDP-star for AP were used in hybridization assay. HBV DNA was detected by autoradiography on a film. The results of 80 samples were compared between the chemiluminescent dot blot hybridization assay with the AlkPhos Direc probe and another assay with the digoxigenin-labelled HBV DNA probe. The correlation of seventy-sample results of fluorescent quantitative HBV DNA PCR assay and dot blot hybridization assay with the AlkPhos Direc probe was analysed. RESULTS: The sensitivity of the AlkPhos Direc probe was 10 pg at least. The coincidence of the AlkPhos Direc probe was 100% compared with that of the digoxigenin-labelled HBV DNA probe. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR assay and dot blot hybridization assay with the AlkPhos Direc probe was 0.98 (P<0.01). CONCLUSIONS: The method detecting HBV DNA in serum with the HBV DNA AlkPhos Direc probe is sensitive and specific. The results of the two assays with the AlkPhos Direc probe or with the digoxigenin-labelled HBV DNA probe are completely coincident. The correlation of HBV DNA quantitative results between fluorescent QPCR assay and dot blot hybridization assay with the AlkPhos Direc probe is satisfactory.

Alkaline Phosphatase↗
PubMed2005

[Genotyping of major outer membrane protein gene of Chlamydia trachomatis by cleavase fragment length polymorphism analysis].

OBJECTIVE: To establish a methed of cleavage fragment length polymorphism (CFLP) analysis with a primer labeled at the 5'-end with digoxigenin for genotyping of Chlamydia trachomatis (Ct). The methods for detection of Ct by major outer membrane protein (MOMP) gene (ompl) with nested polymerase chain reaction (ompl-nPCR) were studied. The incidence of Ct infection in pregnant women, the common genotypes and vertical transmission rate of Ct in Chongqing area during the past one year was also investigated. METHODS: The samples were taken from cervical scrapes of parturient women and nasopharygeal swabs of their neonates from April 2003 to Feb. 2004 in Chongqing Women and Children's Health Care Institute. Totally 300 pairs (605 specimens) were detected by using ompl-nPCR, ompl-PCR (inside pair of primers was used directly) and plasmid-PCR. The results were judged by the modified gold standard (MGS). The ompl-nPCR amplified DNA was purified by recovery of DNA from agarose gel electroelution into dialysis bags. The DNA amplified from ompl-nPCR was sequenced by ABI PRISM 377 DNA sequencer. CFLP assay with a primer labeled at the 5'-end with digoxigenin was created for genotyping of Ct, and was primarily applied. RESULTS: The minimum detectable levels of ompl-nPCR and ompl-PCR corresponded to 2.5 elementary body (EB) and 25 EB, respectively. The sensitivity of ompl-nPCR was 10 times that of ompl-PCR. The positive rate of Ct in the samples from the pregnant women was 11% (33/300). The vertical transmission rate of Ct from mothers to their infants was 24.2% (8/33). The rate of Ct isolated from nasopharyngeal swabs 5 - 10 days after birth was 38.9% (7/18), which was significantly greater than that [3.0% (1/33)] detected within 24 hours after birth (chi(c)(2) = 8.79, P < 0.01). Of the 33 Ct-positive samples from pregnant women, 9 had vaginal delivery and 24 had caesarean section. The vertical transmission rates in vaginal delivery group and caesarean section group were 66.7% (6/9) and 8.3% (2/24), respectively (chi(c)(2) = 9.16, P < 0.01). Incidence of premature rupture of membrane in Ct-positive group was 30.3% (10/33), which was greater than that of Ct-negative groups (13.5%, 36/267, chi(2) = 6.40, P < 0.05). Four different patterns were observed in the 16 Ct-positive samples from 8 pregnant women and 8 matched maternal-infants by using CFLP, which were confirmed by DNA sequencing later. They were type E (3 pairs), type F (2 pairs), type H (2 pairs) and type D (1 pair). Each pair of matched maternal-infantile samples presented identical CFLP pattern. CONCLUSIONS: This study revealed the infection rate of Ct in pregnant women, vertical transmission rate of Ct and the common genotypes of Ct in Chongqing Women and Children's Health Care Institute. The CFLP assay by using a primer labeled at the 5'-end with digoxigenin was first used for genotyping of Ct. The assay showed a good sensitivity and reproducibility, no radioactive contamination, and is simple. Therefore the assay is a potential new method for Ct genotyping.

Cervix Uteri↗
PubMed1990

[Cardioactive contaminants and actual active ingredient content of various digitalis preparations].

To establish possible cardioactive impurities which have not been totally separated while isolating the compound from plant material several preparations containing digoxin (NOVODIGAL = beta-acetyldigoxin, LANITOP = beta-methyldigoxin and LANICOR = digoxin) were analyzed by HPLC-chromatography after careful extraction. Impurities were found (digoxigenin, digoxigenin-monodigitoxosid, digoxigenin-bisdigitoxosid) which on the one hand occur as natural lanatosid-derivates in digitalis leaves but on the other hand also are described as possible metabolites of digoxin in human organism. The experiments have shown that the results of studies in metabolization of digoxin without knowledge of purity of the substance applied have to be interpreted with care.

Digitalis Glycosides↗
PubMed1979

The relationship of age to the metabolism and protein binding of digoxin in sheep.

The relationship of age to the protein binding and metabolism of digoxin was studied in newborn and adult sheep. Protein binding, studied by equilibrium dialysis, varied directly with albumin concentration. The percentage of digoxin bound to protein did not differ with age: 17.2 +/- 1.2% in newborn lambs and 17.6 +/- 1.7% in ewes. The percentage of drug bound did not change significantly over a concentration range of 0.5 to 5 ng/ml. Metabolic studies were performed by thin-layer chromatography on samples of serum, myocardium and urine obtained after animals had been treated for 4 days with [3H]-digoxin. In both newborns and adults, more than 70% of digoxin remained unmetabolized. The mean metabolic composition of all three tissues for newborns and adults, respectively, was: digoxin--80.3%, 77.0%; digoxigenin bisdigitoxoside--9.6%, 12.5%; digoxigenin monodigitoxoside--6.4%, 7.0%; digoxigenin--3.7%, 3.5%. These studies suggest that age-related differences in the effects of digoxin in sheep are not due to variations in drug binding or drug metabolism.

Aging↗
PubMed1994

Detection of human papillomaviruses in paraffin-embedded biopsies of cervical intraepithelial lesions: analysis by immunohistochemistry, in situ hybridization, and the polymerase chain reaction.

One hundred and forty biopsies with an initial diagnosis of cervical intraepithelial lesion (CIL) were tested for the presence of human papillomavirus (HPV) by immunohistochemistry and in situ hybridization using commercial biotinylated probes (Vira-Type in situ assay; Digene Diagnostics, Silver Spring, MD) or probes labeled with digoxigenin by the random primer technique. Immunohistochemistry was more inferior to the in situ hybridization method, with a detection rate of 14% (20/140) compared to 61% (86/140) for the in situ assay with the digoxigenin-labeled probes. Biotinylated probes proved to be slightly less sensitive than digoxigenin-labeled probes, with a detection rate of 53% (74/140). Although less sensitive in our series taken as a whole, immunohistochemistry was positive in a few cases of CILs negative by in situ hybridization, so that the association of these techniques gave the highest detection rate (66%; 92/140). The CILs that remained negative with these methods (34%; 48/140) were investigated by the polymerase chain reaction (PCR) using consensus primers to determine definitively the presence of HPV in these lesions and were reviewed histologically to assess the diagnosis of CILs. The PCR method increased the detection rate of HPV in our series to 76% (107/140). The diagnosis of CILs was confirmed for all the biopsy specimens positive by PCR (15/15; 100%) and for all the HPV negative tissues with histological features of a high-grade lesions (7/7; 100%).(ABSTRACT TRUNCATED AT 250 WORDS)

Female↗
PubMed1993

Development of a DNA probe for identification of bovine herpesvirus 4.

A sensitive and specific DNA probe for detection and identification of bovine herpesvirus 4 (BHV-4) was developed. Cloned fragments from a library of HindIII fragments of the BHV-4 (DN-599) genome were labeled with 32P or digoxigenin and were tested for sensitivity and specificity in detecting viral DNA by dot-blot hybridization. Two probes were identified that detected 10 pg of purified viral DNA, and detected viral DNA in 0.001 micrograms of total DNA extracted from BHV-4-infected cells. Both probes labeled with 32P and 1 labeled with digoxigenin detected viral DNA in samples prepared from cells infected with 2 prototype strains (DN-599 and Movar 33/63) and 4 field isolates of BHV-4. The DNA probes did not hybridize to total DNA prepared from uninfected bovine cells or from cells infected with BHV-1, BHV-2, alcelaphine herpesvirus 1, pseudorabies virus, or equine herpesvirus 1. One probe, labeled with digoxigenin, was tested further by dot-blot hybridization with infected cell lysates that were simply treated with sodium dodecyl sulfate and proteinase K prior to application to the membrane, avoiding extensive DNA purification procedures. This simplified procedure also resulted in specific detection of field isolates of BHV-4 and prototype strains of BHV-4.

Animals↗
PubMed1996

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA.

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.

Cell Line↗
PubMedDate not supplied

Specific primer design and exonuclease III treatment for the reduction of nonspecific staining in direct in situ PCR.

Although direct in situ PCR is more rapid than indirect method for the in situ identification of low copy number genes, several reports indicate serious nonspecific signals with this method. Some procedures have been reported in an effort to eliminate the nonspecific signals, but the results have not been satisfactory. Exonuclease III can progressively digest blunt or recessed 3' termini of double-stranded DNA whereas DNA with 3' overhanging end is resistant to digestion. DNA fragments amplified by PCR with primers incorporating the recognition site for Sph I generate 3' four bases extensions at both end after digestion. These fragments are expected to be resistant to exonuclease III digestion. It is also expected that nonspecifically incorporated digoxigenin would be released by treatment with exonuclease III thereby reducing background. We succeeded in digesting selectively with the samples after standard PCR by Sph I and exonuclease III treatment. However, we failed to eliminate the nonspecific signals of direct in situ PCR. Southern blotting revealed that the amount of nonspecific incorporation was so huge that exonuclease III was unable to release all of nonspecifically incorporated digoxigenin and maintain specific incorporated digoxigenin simultaneously. Direct in situ PCR is a sensitive method. However, its specificity has a significant problem.

Base Sequence↗
PubMed1998

Comparison of isotopic and non-isotopic labelling for in situ hybridisation of various mRNA targets with cRNA probes.

In situ hybridisation methods to localise messenger ribonucleic acid (mRNA) targets in tissue sections or cell preparations using riboprobes can be successful with either isotopic or non-isotopic labelling. Investigators often wish to decide which labelling method provides the maximum specificity, sensitivity and resolution, with minimum nonspecific background. In this study we compared isotopic (35S) and non-isotopic (digoxigenin) labelling, using a variety of probes and paraffin-embedded tissues. The targets were human beta-actin and von Willebrand Factor mRNAs in archival human tissues; and mRNAs for two closely related trefoil factor family (TFF) peptides, TFF2 and TFF3, in rat duodenum. Patterns of localisation with both isotopic and non-isotopic probes were broadly similar for each target. The 35S labelling provided good contrast and sensitive detection under darkfield illumination, but the cellular or subcellular resolution of the target was less precise than that obtained with the digoxigenin-labelled probes in transmitted light. Digoxigenin labelling in individual cells was more clearly demonstrated, but occasionally the contrast of positive staining with background was poor. The sensitivity of each method appeared to be similar for these high-abundance targets, therefore the choice between isotopic and non-isotopic labels is dependent upon the aim of the study and the cellular resolution required.

Animals↗
PubMed2002

Immunoaffinity screening with capillary electrochromatography.

Highly efficient capillary electrochromatographic separations of cardiac glycosides and other steroids are presented. Employing butyl-derivatized silica particles as stationary phase resulted in a nearly three times faster electroosmotic flow (EOF) compared to capillary electrochromatography (CEC) with octadecyl silica particles. On-column focusing with a preconcentration factor of 180 was performed and separation efficiencies of up to 240,000 plates per meter were obtained. Using label-free standard UV absorbance, detection limits of 10-80 nM were reached for all steroids tested. For screening of cardiac glycosides, e.g., digoxin and digitoxin in mixtures of steroids, CEC was combined with immunoaffinity extraction using immobilized polyclonal anti-digoxigenin antibodies and F(ab) fragments. Simply adding small amounts of antibody carrying particles to the samples and comparing chromatograms before and after antibody addition allowed screening for high affinity antigens in mixtures with moderate numbers of compounds. Under conditions of competing antigens, affinity fingerprints of immobilized anti-digoxigenin and anti-digitoxin antibodies were obtained, reflecting the cross-reactivity of eleven steroids. The method provides high selectivity due to the combination of bioaffinity interaction with highly efficient CEC separation and UV detection at several wavelengths in parallel. This selectivity was exploited for the detection of four cardiac glycosides in submicromolar concentrations in an untreated urine sample.

Antibodies↗
PubMed1994

Rapid fluorescence in situ hybridization with repetitive DNA probes: quantification by digital image analysis.

Fluorescence in situ hybridization (FISH) has become an important tool not only in cytogenetic research but also in routine clinical chromosome diagnostics. Here, results of a quantification of fluorescence signals after in situ hybridization with repetitive DNA probes are reported using a non-enzymatic hybridization technique working with a buffer system not containing any formamide or equivalent chemical denaturing agents. Following simultaneous denaturation of both cells and DNA probes, the renaturation time was reduced to less than 30 min. For one of the DNA probes reasonable FISH-signals were even achieved after about 30 s renaturation time. In addition, the number of washing steps was reduced drastically. As a model system, two repetitive DNA probes (pUC 1.77, D15Z1) were hybridized to human metaphase spreads and interphase nuclei obtained from peripheral blood lymphocytes. The probes were labelled with digoxigenin and detected by FITC-anti-digoxigenin. The hybridization time was reduced step by step and the resulting fluorescence signals were examined systematically. For comparison the pUC 1.77 probe was also hybridized according to a FISH protocol containing 50% formamide. By renaturation for 2 h and overnight two FISH signals per nucleus were obtained. Using shorter renaturation times, no detectable FISH signals were observed. Quantification of the FISH signals was performed using a fluorescence microscope equipped with a cooled colour charge coupled device (CCD) camera. Image analysis was made interactively using a commercially available software package running on a PC (80486). For the pUC 1.77 probe the major binding sites (presumptive chromosomes 1) were clearly distinguished from the minor binding sites by means of the integrated fluorescence intensity. For the two (pUC 1.77) or four (D15Z1) brightest spots on the metaphase spreads and in the interphase nuclei hybridized without formamide, integrated fluorescence intensity distributions were measured for different renaturation times (0.5, 15, 30 min). The intra-nuclear variation in the intensity of the two brightest in situ hybridization spots appeared to be slightly higher (CV between 16 and 32%) than the corresponding variation in the metaphase spreads (CV between 10 and 19%). For the D15Z1 probe FISH signals were detected after hybridization without formamide and 15 min and 30 min renaturation. Always four bright spots were visible and tentatively assigned on the metaphase spreads (presumptive chromosome 15 and 9). The intensity variation of each pair of homologues in a metaphase spread showed a CV of 14 or 15%, respectively, for the presumptive chromosome 15, and 8 or 9%, respectively, for the presumptive chromosome 9.

Analog-Digital Conversion↗
PubMed2004

High sensitivity of chemiluminescent methodology for detection of clonal CDR3 sequences in patients with acute lymphoblastic leukemia.

Detection of minimal residual disease (MRD) in patients with B-cell acute lymphoblastic leukemia (B-ALL) has been achieved using several radioactive labelling methodologies; however, limited information exists about the use of chemiluminescent labelling. Although many malignant disorders are related to cytogenetic alterations, there is not a consistent chromosomal translocation that could serve as a tumour marker for the monitoring of MRD. ALL are derived from B-lymphocytes in 80% of cases. In the early stages of their maturation, the immunoglobulin heavy chain genes (IgH) undergo rearrangements among their V, D, and J segments, giving rise to the Complementary Determining Regions (CDR). Among these, CDR3 is considered unique for each lymphocyte and used as a tumour-specific marker in B-ALL patients. In this study, the CDR3 was labelled with digoxigenin and used as a patient-specific probe to test its sensitivity for further detection of MRD. Fourteen pretreatment samples of bone marrow (BM) or peripheral blood (PB) from B-ALL patients were included. Tumour-specific probes were designed from each clonal product by elimination of the consensus sequences. Ten digoxigenin-labelled probes were hybridized with a mixture of their respective clonal DNA and the polyclonal product from a normal healthy donor, in serial dilutions from 1:1 up to 1:10(7). A sensitivity range of 1:10(3)-1:10(6) was obtained, with an average of 1:10(5). Crossed tests performed in four patients, showed right probe specificity in all cases. We propose that the design of allele-specific probes with chemiluminescent labelling, represents a reliable, sure and sensitive alternative methodology for MRD detection in patients with B-cell lymphoproliferative disorders.

Alleles↗
PubMed2001

Detection of adeno-associated virus DNA in female genital samples by PCR-ELISA.

Adeno-associated viruses (AAV) are human parvoviruses that require helper function for their replication. Several studies have demonstrated that AAV DNA sequences can be found in the female genital tract but the incidence of infection seems very variable. A PCR-ELISA method detecting AAV DNA was developed for combining the specificity and the sensitivity of conventional PCR with an objective interpretation of the results. In the PCR-ELISA, a defined number of cells from cervical specimens were digested and amplified with concomitant digoxigenin labeling. Digoxigenin-labeled amplified products hybridized to a specific biotinylated probe were captured in streptavidin-coated microtiter wells by a biotin-streptavidin binding and were visualized by colorimetric immunoenzymatic reaction. PCR-ELISA was carried out in 110 cervical cytological specimens of women with or without the concomitant detectable presence of papillomavirus (HPV) DNA and the results were compared with those obtained by conventional PCR followed by dot blot hybridization. When compared to conventional PCR considered as reference standard, PCR-ELISA was found to be 98% sensitive and 96% specific. Out of the total 110 samples examined, 52.7% were positive for AAV DNA by both techniques, demonstrating a high prevalence of AAV infection in the uterine cervix. When analyzing samples with or without the presence of HPV DNA, 63.2 % of the samples were positive for HPV DNA and 41.5% of the samples were negative for HPV proved positive for AAV DNA by both PCR-ELISA and conventional PCR. Hence, PCR-ELISA, which can be completed in 1 day, proved to be a reliable method for an objective detection of AAV DNA in clinical samples. The present study showed a frequent infection of the cervical epithelium with AAV both in the presence and absence of HPV infection.

Cervix Uteri↗
PubMed1993

Synthesis of PCR-derived, single-stranded DNA probes suitable for in situ hybridization.

We report the novel synthesis of polymerase chain reaction (PCR)-derived single-stranded DNA (ssDNA) probes and their subsequent application in in situ hybridizations. Serial transverse sections of an 11.5-day postcoitum mouse embryo were hybridized to a 33P-ssDNA, 33P-RNA, or 35S-RNA probe corresponding to the same 181-bp sequence in the myogenin cDNA. Signal obtained using 33P-ssDNA was more intense than that using 33P-RNA probe, while signal/noise ratios obtained with both 33P-probes were far superior to those obtained with 35S-probe. Digoxigenin-labeled chicken growth hormone (GH) ssDNA gave slightly more intense signal than did digoxigenin-labeled chicken GH RNA when hybridized to chicken pituitary sections. 32P-ssDNA probes were found to be suitable for Northern blot hybridization. Advantages of using ssDNA probes for in situ hybridization include: (1) The ssDNA technique is rapid and simple. There was no need to clone a DNA template into a special RNA vector or order special T7-containing PCR primers. ssDNA probes can be synthesized in less than 1 day using any primers which currently exist in a laboratory (optimal probe length for in situ hybridization is between 50 and 200 bp). (2) In three separate in situ experiments, ssDNA probes yielded more intense signal than RNA probes. (3) ssDNA probes are potentially more stable than RNA probes. (4) Since the RNAse rinse is eliminated, posthybridization rinses are shortened when hybridizing with ssDNA probes. The ssDNA probes produced by this protocol can be labeled with a variety of different isotopes (both radioactive and nonradioactive), and are excellent probes for use in in situ hybridizations.

Animals↗
PubMed1993

Quantitative measurement of nonisotopically labeled polymerase chain reaction product.

A novel approach for evaluation of polymerase chain reaction product was described. A biotin-labeled DNA (or RNA) probe is hybridized to the digoxigenin-labeled target PCR product. The hybrids are retained on an anti-biotin antibody-coated plate. An anti-digoxigenin Fab conjugated with alkaline phosphatase is used for generating either a fluorescence or a chromagenic substance. Both fluorogenic and chromagenic systems can be used for quantitative measurement of the PCR product/probe hybrids. The method is specific, fast, nonisotopic, sensitive, quantitative, and practical.

Base Sequence↗
PubMed1998

Carbohydrate-based probes for detection of cellular lectins.

Carbohydrate (spacered saccharide residue, Glyc) probes with various tags were synthesized as analytical tools for study of cellular lectins, i.e., Glyc-polyacrylamide-3H, Glyc-PAA-biotin, Glyc-PAA-fluorescein (flu), and Glyc-PAA-digoxigenin, where PAA is a soluble polyacrylamide carrier of approximately 30 kDa. Binding of all types of probes, where Glyc is the sialyl Lewis X (SiaLeX) tetrasaccharide or a blank saccharide, was assessed using Chinese hamster ovary (CHO) cells either transfected with the E-selectin cDNA or mock-transfected. High binding of SiaLeX-PAA-3H to E-selectin-transfected cells and absence of binding to control cells (both native and permeabilized) allowed the conclusion that the polyacrylamide carrier and the spacer arm do not contribute significantly to the binding. The biotinylated probe showed a high level of nonspecific binding in cell enzyme-linked assays. A similarly built digoxigenin-labeled probe was significantly better. In flow cytometry assays, the fluorescein probe demonstrated a specific binding to E-selectin-transfected cells of a similar level to that given by an anti-E-selectin antibody. In addition, it could be inhibited by the anti-E-selectin antibody, further demonstrating specificity. Tumors were obtained from nude mice by injection of CHO E-selectin or mock-transfected cells. The fluorescent SiaLeX-PAA-flu probe could bind to tumor sections from E-selectin-positive CHO cells, but not from control CHO cells. These probes can thus be used to reveal specifically complex carbohydrate-binding sites on cells either in culture or on tissue sections.

Animals↗
PubMed2001

Transcript quantification based on chemical labeling of RNA associated with fluorescent detection.

A general method for RNA measurement, based on chemical labeling of RNA with digoxigenin (without retrotranscription), has been established. Labeled RNA is hybridized with nylon membranes containing spot blots of PCR-amplified gene fragments and the fluorescence detection is mediated via specific anti-digoxigenin antibody coupled to alkaline phosphatase. The method was optimized in order to be quantitative, and high precision (less than 24% error) was obtained, allowing analysis of relatively small changes in gene expression. When the quantity of cellular RNA used in this method is maintained constant and the amount of RNA in the cell determined, the true intracellular transcript concentrations can be determined, rather than simple abundance of a messenger in RNA population. This RNA quantification technique was extended to macroarrays blotted automatically and the validity of the method was tested by comparison with expression data obtained by Northern blotting.

Blotting, Northern↗