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At least 649 records · Page 36Linked to original sources
PubMed1994

Direct detection of insulin-like growth factor II (IGF-II) by chemiluminescence without interference by IGF binding proteins.

A dot blot method for the detection of picogram quantities of human and rat insulin-like growth factor II (IGF-II) in serum-free conditioned media is described. The crossreactivity of human recombinant IGF-I in the assay was < 10%. None of the IGF binding proteins (IGFBP 1-6) diminished the IGF-II signal. In contrast, significant interference by the IGFBPs was observed when the same concentrations of IGFBPs and 125I-IGF-II were used in a radioimmunoassay which utilized the same antibody. Why IGF-II is detected in the dot blot assay without IGFBP interference is not understood. We speculate that the conformation of the IGF-II/binding protein complex may be altered by binding to the nitrocellulose, exposing the IGF-II epitope that is recognized by the antibody. IGF-II was detected in 1 microliter of serum-free conditioned media from BRL 3A cells (which secrete IGF-II) while no signal was generated by 50 microliters of BRL 3A2 conditioned media (which do not secrete IGF-II). In summary, this method is ideal for screening cells in serum free-culture for production of IGF-II without the need for separation of IGF-II from cell derived IGFBPs.

Animals↗
PubMed1997

Direct detection of Campylobacter jejuni in chicken cecal contents by PCR.

Campylobacter jejuni in chicken feces was detected by PCR and Southern blot hybridization (SBH). The detection limits of C. jejuni in chicken feces were 34,000 cells by PCR and 340 cells by SBH. Some cecal contents of chickens up to 3 weeks old were C. jejuni positive by SBH whereas all of them were negative by PCR. Two of 51 cecal contents of 18-day-old chicken embryos were C. jejuni positive by PCR and SBH; but, C. jejuni were not isolated from the samples by conventional culture with selective enrichment.

Aging↗
PubMed2006

Direct detection of herceptin/trastuzumab binding on breast tissue sections.

The protooncogene product HER-2/neu is the target of the humanized monoclonal antibody trastuzumab (Herceptin). Several tests are used clinically to identify patients with HER-2/neu overexpression based on evaluation by pathologists of gene amplification by fluorescence in situ hybridization or protein expression using immunohistochemistry (IHC). A simple technique has been developed for staining formalin-fixed, paraffin-embedded breast cancer tissue using unmodified Herceptin/trastuzumab as the primary antibody. Results were compared with staining with the commercial kit, HercepTest, as well as with polyclonal anti-HER-2/neu antibodies and with biotinylated trastuzumab. These procedures were tested using four breast cancer microarrays. There were 854 cores that were stained with all four antibodies, representing 325 cases. A standard 4-point scoring system (0-3) was used. A total of 156 cases (48%) were scored as 0 by all the methods used and 31 (9.5%) were positive (3+) by all methods. Of interest, three cases scored negative using polyclonal anti-HER-2/neu antibodies but were positive using unmodified trastuzumab. To clarify this discrepancy, whole sections of tumors were examined with both antibodies using double labeling. There were some tumors that demonstrated a mosaic pattern of staining with neighboring cells or groups of cells stained exclusively with one antibody or the other. These results demonstrate that unmodified humanized or human therapeutic antibodies could be used for preclinical testing or in a clinical laboratory setting for IHC-based selection of patients for treatment, and results of such selection could be different from those obtained using polyclonal antibody-based IHC procedure.

Antibodies, Monoclonal↗
PubMed1994

Direct detection of Porphyromonas gingivalis in Macaca fascicularis dental plaque samples using an oligonucleotide probe.

Oligonucleotide probes complementary to the hypervariable regions of the 16S rRNA of Porphyromonas gingivalis, and previously shown to specifically identify human P. gingivalis strains to the species level, were tested for their ability to recognize P. gingivalis from nonhuman primates (Macaca fascicularis), either as distinct isolates or in subgingival dental plaque. The 32P-labeled probes hybridized with all 147 monkey isolates identified as P. gingivalis by morphology and biochemistry, but did not hybridize with any of the 331 isolates representing 17 genera of bacteria unrelated to P. gingivalis, or to the more closely related P. endodontalis and P. asaccharolytica. This corresponds to sensitivities and specificities of 100%. Of 76 M. fascicularis plaque samples, P. gingivalis was detected by probe and culture in 67. Of 26 human plaque samples taken from separate individuals free of periodontal disease, 23 failed to demonstrate P. gingivalis by probe or culture. The results of the combined 102 monkey and human plaque samples indicate that, when compared to culture as the "gold standard," the P. gingivalis probe had a sensitivity of 96%, a specificity of 87%, and an overall agreement with culture of 93%. These results reveal that the oligonucleotide probes used to identify P. gingivalis are specific for this organism, and give results comparable to culture methods for detecting the presence of P. gingivalis in M. fascicularis dental plaque.

Animals↗
PubMed2006

Direct detection of bacterial faecal indicators in water samples using PCR.

The presence of enteric pathogens in water resources represents a serious risk for public health. Therefore, their precise detection, and especially detection of E. coli, which is obviously regarded as the main indicator of faecal contamination of water, is an essential step in ensuring bacterial safety of water. Numerous PCR protocols for detection of E. coli have been published to date. They are usually based on amplification of regions derived from lacZ (beta-D-galactosidase) and uidA (beta-D-glucuronidase) gene sequences. However, these methods are not universal enough for precise detection of all E. coli strains found in water samples. We developed a novel triplex PCR method for detection of E. coli in which cyd gene coding for cytochrome bd complex was co-amplified along with lacZ and uidA genes. Our triplex PCR approach significantly increases the specificity and reliability of E. coli detection in water samples. This approach allowed us to distinguish Shigella flexneri from E. coli. In addition, we were able to detect even non-coliform Klebsiella and Raoutella spp., some of which can also cause infections to humans.

Base Sequence↗
PubMed1996

RNA amplification for direct detection of mycobacterium tuberculosis in respiratory samples.

A target amplified test system (AMTDT, Gen-Probe, San Diego, California, USA) was compared with conventional standard methods in the detection of Mycobacterium tuberculosis in sputum and other respiratory specimens from patients with suspected tuberculosis. The highest incidence of positive samples was seen with the AMTDT test. Out of 450 samples from patients with known or suspected tuberculosis, 43 (9.6%) were positive by culture, and 47 (10.4%) with AMTDT. After discrepancy analysis, 7 of the culture-negative but AMTDT-positive samples were judged to be true positive, giving the AMTDT a sensitivity of 92.0% and a specificity of 99.8%. The AMTDT test was rapid, easy to perform, sensitive, and highly specific.

Bronchoalveolar Lavage Fluid↗
PubMed1980

Direct detection of the vasopressin precursor.

Cysteine-rich proteins were isolated from the hypothalamo-neurohypophyseal tract of dogs by high performance molecular weight chromatography. Trypsin digestion of these proteins produced a low molecular weight (LMW) peptide which was identified, by chemical and immunological assays, as (Arg8) vasopressin. There appear to be two forms of the precursor protein, one which has vasopressin immunoreactivity, and one which does not. Trypsin digestion of this latter protein produces high as well as LMW immunoreactivity. This suggests that the non-immunoreactive protein may be the precursor to the immunoreactive protein.

Animals↗
PubMed1989

Direct detection of peroxyl radicals formed in the reactions of metmyoglobin and methaemoglobin with t-butyl hydroperoxide.

The reaction of metmyoglobin and methaemoglobin with t-butyl hydroperoxide has been investigated using stopped-flow e.s.r. spectroscopy. The major species observed immediately after mixing has been identified as the basis of its g value (2.014) and line width as being due to a peroxyl radical. The detection of this species under anaerobic conditions suggests that this species is the t-butyl peroxyl radical rather than a secondary species. The immediate observation of this species on mixing suggests that this species is arising via a reaction involving the intact haem moiety rather than via 'free iron' produced via oxidative damage. Detection of a second, oxygen-dependent, species, which is formed at a slower rate, in the metmyoglobin reaction suggests that the initial peroxyl radical subsequently reacts with the protein to give a protein-derived radical.

Chemical Phenomena↗
PubMed1998

One step direct detection of recurrent mutations in the breast cancer susceptibility gene, BRCA1.

Germ-line mutations of the BRCA1 gene have been linked to 85% of hereditary breast and ovarian cancers. More than one hundred mutations have been reported, including several which are over represented within the Ashkenazi Jewish (185delAG) and Irish families (1294del40). These recurrent mutations are cost-effective targets for presymptomatic screening of cancer susceptibility. Most current techniques such as single strand conformation polymorphism, heteroduplex analysis, DNA sequencing, and protein translation termination assays are multistep, time consuming methods and require radioactive isotopes for mutation detection. As an alternative, we have developed a single step, non-radioactive allele specific oligonucleotide (ASO) PCR assay designed to target any known mutation. Its application to detection of the BRCA1 185delAG and 1294del40 mutations is described here. The ASO PCR is efficient, highly sensitive, non-radioactive, specific for individual mutations, performed in a single step, and is amenable to large-scale screening for other known mutations.

Alleles↗