Secondary Detection System Research Articles

Generation of a Biotin-Tagged Dual-Display Phage.

Phage display is widely used in biomedical research. One of the great advantages of phage display is the specificity of the connection of a foreign peptide exposed outside the capsid to the intended target. Secondary detection systems, which are often laborious and costly, are required to identify and quantify the peptide/target interaction. In this study, we generated a novel dual-display phage to facilitate the detection and quantification of the peptide/target interaction. First, we generated a biotin-tagged phage by adding a small biotin-accepting peptide (sBT) to gene-3 of the M13K07 helper phage. Subsequently, we enhanced the M13K07 biotin-tagged phage by incorporating a selective peptide on gene-8, which is then exposed to the phage capsid. The exposed peptide acts as a probe to bind to a selective molecular target, whose interaction can be readily visualized thanks to the biotinylated phage. Our versatile dual-display phage exhibits high flexibility; by swapping the displayed peptide/probe, one can change the phage target while retaining the sBT gene in-frame with the pIII. We expect the generated biotin-tagged dual phages to be used as a multifunctional probe to couple with several streptavidin-biotin-based systems.

Laser generated Ultrasound System (LUS) and its application for the Non-Destructive Testing of metallic components

One of the investigation areas regarding the Ultrasonic Non-Destructive Test (UT-NDT) is the development of non-contact technologies like Air-Coupled UT, EMAT or UT generated by Laser. These technologies show several interesting advantages like no couplant needed or their application to the inspection of components with complex geometries and extreme surface conditions (temperature, roughness, radiation…). In the Laser Ultrasonics Techniques (LUS), ultrasound is generated by a pulsed laser that produces a thermal expansion of the surface where it impacts and generates vibrations at ultrasonic frequencies. These vibrations can travel through the inside of the material and its surface; and they are reflected, refracted or diffracted in the defects or discontinuities of the material. The disturbances generated by the interaction of the vibration with the defects are detected in another point of the material using a secondary detection system. Tecnatom is working with LUS technology since 2010, acquiring, installing and optimizing a laboratory system for its implementation in the inspection of metallic materials (aluminum, carbon steel and stainless steel). During this time, several works have been carried out to determine the scope of the technology as: initial study for the characterization of the waves generated by the laser; studies in test blocks without discontinuities to determine the experimental conditions in which ultrasonic disturbances are generated in thermo-elastic and ablative regime; simulations and microstructural studies of the pieces to determine both the structural state and the residual stresses caused by the incidence of the laser; inspection of blocks with artificial reflectors, both superficial and embedded, and set-up techniques to increase the amplitude of the detected signal; optimization of the detection system through the combination of technologies and identification of safety criteria, use and improvement of the system for validation use in industrial environments. In this work, Tecnatom presents the main conclusions of the development carried out to determine the capacities and limitations of the techniques based on Laser Ultrasonics for its application in Non-Destructive Testing for the inspection of metallic components.

Morphological study of the retina of WAG/Rij rats with pigmentary degeneration in postnatal ontogenesis

The aim of the study was to detect morphofunctional features of the retina of WAG/Rij rats during postnatal development from the 1st to the 360th day after birth.Material and methods. The study included retina of the inbred WAG/Rij rats (60 eyeballs from 30 rats totally) from the 1st to the 360th day of life. Standard histological studies were performed on paraffin sections stained with hematoxylin and eosin. Immunohistochemical method was used to determine the expression of acidic glial fibrillar protein GFAP in the rat retina. Mouse monoclonal antibodies (Santa Cruz Biotechnology) and a universal secondary detection system (NovocastraTM) were used for imaging. The degree of protein expression in the retina of WAG/Rij rats was compared in different age groups.Results. It was found that after birth, the retina of rat models of the WAG/Rij line is formed in the same way as the retina of rats of other strains and acquires a definitive structure only by the end of the second week (correlates with the opening of the eyes). On the 20th day, the first signs of dystrophic and destructive processes appear in the retina of WAG / Rij rats progressing as they grow older and leading to retinal gliosis. The increase in the expression of acidic glial fibrillar protein GFAP begins from the 30th day and increases with age as destructive processes in the retina increase. Conclusion. The early postnatal development of the retina of the WAG/Rij rats, which correlates in character with the postnatal retina development of rats of other strains, is interrupted by the launch of destructive processes in the retina soon after its complete differentiation. The further intensifying cascade of degeneration over time leads to the death of retinal neurons and their replacement by glial cells.

Differential reactivity of serum immunoglobulins from Brazilian wild mammals to staphylococcal A and streptococcal G proteins

Human pathogens have evolved to infect vertebrate hosts other than human beings without causing symptoms of the disease, thus permitting them to complete their life cycle and to develop into infectious forms. The identification and management of infected animals are alternatives to control dissemination of the disease and to prevent human illness. In the current study, the potential use of staphylococcal A or streptococcal G proteins was evaluated with enzyme-linked immunosorbent assays (ELISAs) for seroepidemiological studies. Sera were collected from animals that were representative of 23 different Brazilian wild mammals. A high protein A binding rate was observed in all animals, except for the orders Didelphimorphia, Artiodactyla, and Rodentia, in which affinity was medium or low. Affinity for streptococcal G protein was higher in animals of the order Artiodactyla, whereas no streptococcal G protein binding was observed in samples obtained from felines (order Carnivora). Bacterial protein binding to mammalian immunoglobulins was confirmed by immunoblotting. The results suggest that secondary detection systems should be better investigated in ELISA protocols before their implementation in seroepidemiological studies involving wild mammals.

Homozygous Mutations in ADAMTS10 and ADAMTS17 Cause Lenticular Myopia, Ectopia Lentis, Glaucoma, Spherophakia, and Short Stature

Weill-Marchesani syndrome (WMS) is a well-characterized disorder in which patients develop eye and skeletal abnormalities. Autosomal-recessive and autosomal-dominant forms of WMS are caused by mutations in ADAMTS10 and FBN1 genes, respectively. Here we report on 13 patients from seven unrelated families from the Arabian Peninsula. These patients have a constellation of features that fall within the WMS spectrum and follow an autosomal-recessive mode of inheritance. Individuals who came from two families and met the diagnostic criteria for WMS were each found to have a different homozygous missense mutation in ADAMTS10. Linkage analysis and direct sequencing of candidate genes in another two families and a sporadic case with phenotypes best described as WMS-like led to the identification of three homozygous mutations in the closely related ADAMTS17 gene. Our clinical and genetic findings suggest that ADAMTS17 plays a role in crystalline lens zonules and connective tissue formation and that mutations in ADAMTS17 are sufficient to produce some of the main features typically described in WMS.

Antibody Elution Method for Multiple Immunohistochemistry on Primary Antibodies Raised in the Same Species and of the Same Subtype

Double or multiple antigen labeling in IHC classically relies on the existence of primary antibodies raised in different species or of different IgG isotypes to ensure the specific labeling with the secondary detection systems. However, suitable pairs of primary antibodies are not always available or the best choice (e.g., as diagnostic tools). During the last few years, several methods have been proposed to overcome this, but none of them offers the flexibility needed for reliable double or multiple enzymatic or fluorescent IHC. We present here a procedure that elutes the antibodies after a first round of immunolabeling, which, in combination with precipitation-based detection systems, allows multiple IHC rounds even for primary antibodies raised in the same species and IgG isotype. Compared with other proposed methods, this procedure ensures a reliable enzymatic or fluorescent staining without cross-reactivity and without loss of tissue antigenicity, thus offering a flexible tool for colocalization studies and pathological diagnosis. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

TTF-1 Expression in Ovarian and Uterine Epithelial Neoplasia and its Potential Significance, an Immunohistochemical Assessment With Multiple Monoclonal Antibodies and Different Secondary Detection Systems

Thyroid transcription factor-1 (TTF-1) is a 38-kd homeodomain containing DNA-binding protein, identified in thyroid and lung as a regulator of thyroid-specific genes and surfactant and Clara cell secretory protein gene expression. TTF-1 has been used as a reliable lineage marker for lung adenocarcinoma and thyroid carcinoma in surgical pathology. However, TTF-1 expression has been recently reported in carcinomas of other origins including female genital tract. We evaluated TTF-1 expression with 3 primary different antibodies (8G7G3/1, SPT24, and BGX-397A) and 2 secondary automated detection systems (Envision+/Dako autostainer versus Refine/Bond Max) in 104 ovarian and endometrial tumors on routine surgical specimens and 108 ovarian tumors on tissue microarray (TMA) specimens. SPT24 and Refine/Bond Max autostainer was the most sensitive system among the primary antibodies and secondary detection/autostainers tested. By using SPT24/Refine/Bond Max, TTF-1 reactivity could be detected in all major histologic subtypes of gynecologic tumors and up to 26% of all cases tested on routine surgical specimens and 6.4% on TMA. TTF-1 was most frequently detected in uterine malignant mixed Müllerian tumor (82%), more common in uterine tumors than ovarian tumors, and more common in surgical specimen than TMA. When present, tumor cells can be rarely positive or diffusely positive for TTF-1 reactivity. In addition to malignant tumors, TTF-1 was also detected in benign tumors and benign tubal and endometrial epithelia. TTF 1 immunostaining has the potential to misguide a pathologist to conclude an ovarian or endometrial tumor being a lung metastasis. However, the role of TTF-1 in female genital tract and its tumors is unknown.

Polymerase Chain Reaction in Intraocular Inflammation

Polymerase chain reaction (PCR) is a technique involving enzymatic amplification of nucleic acid sequences in repeated cycles of denaturation, oligonucleotide annealing and DNA polymerase extension. It is a powerful molecular biologic tool that allows the rapid production of analytic quantities of DNA from small amounts of starting material. PCR can be performed on nearly any ocular specimen or biopsy. For diagnosis of uveitis, the obtained sample is usually an anterior chamber paracentesis or vitreous tap. PCR potentially is more sensitive than culture for detection of many organisms. By utilizing a secondary detection system in concert with the initial PCR reaction, perfect specificity can be assured. The initial application of PCR diagnostics to ophthalmic disease was in the detection of viral uveitis. PCR has also been implicated in studies of noninfectious uveitis. The most common application is HLA typing. A universal bacterial PCR can be very helpful for the diagnosis of bacterial endophthalmitis at an early stage of the disease.

Human Breast Fibroblasts Inhibit Growth of the MCF10AT Xenograft Model of Proliferative Breast Disease

Stromal fibroblasts are important for normal breast homeostasis and regulation of epithelial growth; however, this regulatory function is altered during carcinogenesis. To study the role of fibroblasts in the development of breast cancer, fibroblasts derived from normal breast (NAFs) were incorporated into the MCF10AT xenograft model of progressive proliferative breast disease. The persistence of human NAFs in xenografts was established by intracellular labeling and tyramide-coupled fluorescent in situ hybridization. Overall, the number of MCF10AT epithelial structures was decreased, and the rate of epithelial cell apoptosis was increased in xenografts containing NAFs. However, these changes were primarily in low-grade epithelial structures, corresponding to normal or mildly hyperplastic ductal epithelium. The level and rate of apoptosis of high-grade epithelial structures, corresponding to in situ and invasive carcinoma, were not consistently altered by NAFs. In addition, there was variability in the growth-inhibitory capacity of NAFs derived from different individuals. NAFs induced changes in the morphology of high-grade MCF10AT structures and in xenograft stroma, including the composition of extracellular matrix, and increased angiogenesis and lymphocytic infiltration. These findings imply that NAFs can inhibit the growth of normal and hyperplastic epi-thelium but are less able to regulate the more transformed epithelial cells that arise during carcino-genesis.

A modified quantitative EMSA and its application in the study of RNA–protein interactions

Electrophoretic mobility shift assay (EMSA) is used to detect the complex of protein and nonradioisotope-labeled probe qualitatively. In this paper, we describe a modified quantitative EMSA, which uses biotin-labeled RNA in the complex formation. The RNA–protein complex is separated by agarose gel electrophoresis and capillary transferred to a positively charged nylon membrane. It is then detected through a secondary detection system using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT) as the colorimetric precipitating substrate. After scanning and quantification by an image analysis program, ImageQuant, it was observed that the optical density of the bands was proportional to the decadic logarithm value of standard RNA quantities in the tested range. By using the standard curve of the optical densities plotted against the logarithm values of RNA quantities in the linear range, we can calculate RNA quantities according to the optical density of the band and make a quantitative analysis of EMSA. This method was applied successfully in the study of the interactions between the AU-rich element (ARE) and HuR, which is one of the human members of the (embryonic lethal abnormal vision) ELAV family. The results reveal that the biotin-labeled AUFL transcripts in the RNA–protein complex can be detected quantitatively, while maintaining the biological features of its binding ability to the HuR protein. By this method, it is possible to conduct qualitative and quantitative analyses of the EMSA in the study of RNA–protein interactions.

Identification of repertoires of surface antigens on leukemias using an antibody microarray.

We have previously described a microarray of cluster of differentiation (CD) antibodies that enables concurrent determination of more than 60 CD antigens on leukocytes. This procedure does not require protein purification or labeling, or a secondary detection system. Whole cells are captured by a microarray of 10 nL antibody dots immobilized on a nitrocellulose film on a microscope slide. Distinct patterns of cell binding are observed for different leukemias or lymphomas. These haematological malignancies arise from precursor cells of T- or B-lymphocytic, or myeloid lineages of hematopoiesis. The dot patterns obtained from patients are distinct from those of peripheral blood leukocytes from normal subjects. This microarray technology has recently undergone a number of refinements. The microarray now contains more CD antibodies, and a scanner for imaging dot patterns and software for data analysis provide an extensive immunophenotype sufficient for diagnosis of common leukemias. The technology is being evaluated for diagnosis of leukemias with parallel use of conventional diagnostic criteria.

High-resolution immunocytochemical labeling of replicas with ultrasmall gold.

The use of 10-15-nm gold probes in freeze-fracture immunocytochemistry sometimes results in poor immunogold labeling. Replica sites are labeled with only one or two gold particles, making it unlikely that the labeling depicts the true distribution of antigen. In this study, the feasibility of using ultrasmall ( approximately 1.4-nm) gold probes for immunocytochemical labeling of replicas was examined. When HLA Class I in neutrophil membrane replicas was labeled with various sized immunogold particles as the secondary detection system, the apparent distribution density was inversely related to the size of the particles (1.4-nm > 5-nm >10-nm >15-nm). Indeed, the density of the apparent distribution of HLA Class I labeled with 1.4-nm gold particles was about sevenfold greater than when labeling was carried out with the 10-nm gold particles. Similar results were obtained with CD16, another neutrophil membrane protein. Silver enhancement was required to visualize the 1.4-nm gold particles, but this procedure did not adversely affect replica membranes. These results suggest that, when followed by silver enhancement, 1.4-nm gold particles are effective probes for achieving high-resolution immunocytochemical labeling of replicas.(J Histochem Cytochem 47:569-573, 1999)

Apoptosis During Diploid Parthenogenesis and Early Somatic Embryogenesis of Norway Spruce

In diploid parthenogenesis of Norway spruce, specific nuclei were ontogenetically eliminated in cells programmed for cell death or apoptosis. Nuclear elimination was defined morphologically and biochemically for the degenerating ventral canal nucleus of egg-equivalents, and in differentiating suspensors of the axial tier of early embryos. Nuclei became pycnotic and disintegrated with the release of nucleoli and nuclear fragments into the cytoplasm. Enucleation in tubular cells of the axial tier was preceded by a reduction in the number of interphase nucleoli. Apoptosis was biochemically characterized by the activation of endonuclease activity that contributed to massive morphological changes in nuclei and ultimately to cell death. Endonuclease activity in individual nuclei was determined by a terminal deoxynucleotidyl transferase (TdT). This enzyme labeled the 3'OH ends of DNA, generated by DNA nicking with biotin-conjugated dUTP, for visualization with a secondary detection system using a colorimetric su...

A fast highly sensitive colorimetric enzyme immunoassay system demonstrating benefits of enzyme amplification in clinical chemistry

A method for greatly enhancing the sensitivity of assays employing enzyme labels is described which offers advantages in assays for a wide range of analytes. The principle of the new approach is that the enzyme label gives rise to a catalytic activator for a specific secondary detection system, the activity of which is measured and related back to the amount of label present and thus of the analyte it is being used to determine (C.H. Self, Eur. Pat. Appl. 80303478.4, 15.4.81 exclusively licenced to IQ (Bio) Ltd.). The general principle of enzyme amplification is illustrated by using alkaline phosphatase as the labelling enzyme and nicotinamide adenine dinucleotide phosphate (NADP) as its substrate. The nicotinamide adenine dinucleotide (NAD) formed catalytically activates a strictly NAD specific redox cycle which produces a coloured formazan as the end product. The measured absorbance is at least two orders of magnitude greater than that achieved by conventional methods. The application of this method to immunoassay is demonstrated by a sensitive, rapid and precise assay for human prostatic acid phosphatase (PAP). Some of the many other applications of this methodology are discussed.

Quantitation of ciprofloxacin in body fluids by high-pressure liquid chromatography.

We describe a reverse-phase high-pressure liquid chromatography method for the quantitation of a new quinoline carboxylic acid antimicrobial agent, ciprofloxacin (Bay o 9867). This assay utilizes the intrinsic fluorescence of ciprofloxacin for primary detection but employs UV absorption as a secondary detection system. Mobile phases contained methanol and phosphate buffer and used a common C18 mu Bondapak column. A single precipitation step of a 50-microliter specimen was the only sample preparation necessary. The assay is linear from 2,000 to 10 ng/ml and sensitive to 5 ng/ml. The mean recovery of ciprofloxacin from serum was 105.7%. The coefficient of variation was less than or equal to 3.1% for same-day precision and less than or equal to 6.3% for assay-to-assay precision. Because the assay requires only small specimen volumes and minimal sample preparation and because of its defined characteristics, this assay would be ideal for clinical trials and pharmacokinetics studies of ciprofloxacin.