Browsing by Subject "Antibodies"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Publication
    Open Access
    Epithelial lung cell marker, current tools for cell typing
    (Murcia : F. Hernández, 1994) Kasper, M.; Singh, G.
    This review discusses current irnmunohistochemical and lectin histochemical approaches to identify and to distinguish the different epithelial cell populations of the pulmonary tissue. Special emphasis is given to the characterization of pulmonary alveolar and bronchial epithelial cells and mesothelial cells. Structural proteins, membrane molecules and secretory products of the alveolar epithelium, which have already been characterized and which may be useful for monitoring the developmental or pathological processes, are listed and briefly described.
  • Thumbnail Image
    Publication
    Open Access
    General insufficiency of the classical CDC-based crossmatch to detect donor-specific anti-HLA antibodies leading to invalid results under recipients’ medical treatment or underlying diseases
    (F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2012) Schlaf, Gerald; Mauz-Körholz, Christine; Ott, Undine; Leike, Steffen; Altermann, Wolfgang
    Antibodies directed against HLA antigen of a given donor represent the most prominent cause for hiper-acute and acute rejections. In order to select recipients without donor-specific antibodies the complement-dependent cytotoxicity (CDC-) crossmatch as the standard procedure was established. As a functional assay it strongly depends on the availability of isolated donor lymphocytes and in particular on their vitality. However, due to several diseases or pharmacological treatment of a given recipient unexpected “false-positive” results of the CDC- crossmatch may arise. We here present three groups of patients which demonstrate the limits of the conventional crossmatch. 1) Kidney recipients before living donations exhibited positive CDC-reactions due to their conditioning using the therapeutical anti-CD20 mAb Rituximab (n=7), routinely used to deplete B-cells, or the anti-CD25 mAb Basiliximab (n=2) to inhibit the proliferation of activated T-cells. 2) Recipients suffering from various leukaemias (n=5) exhibited “positive” CDC-crossmatches using PBL of the donors, although formerly these patients had never shown anti-HLA antibodies. Instead of donor-specific allo-antibodies, cytostatic agents such as 6-Mercaptopurine led to an unspecific cell death. 3) Patients projected for post mortem or living kidney donations (n=44) exhibited “positive” CDC-crossmatch results which were not in accordance with their former antibody status and, partially, with high degrees of HLA-matching. These implausible results were due to underlying auto-immune false-positive B-cell crossmatches by immune diseases, mainly of the systemic Inmune Complex Type III such a Lupus Erythematosus, mainly leading to false-psitive B- cell crossmatches by complexes binding to Fcγ -receptors. In all these 58 cases the alternatively performed ELISA-based “Antibody Monitoring System” (AMS-) crossmatch assay was not artifically affected, suggesting that this assay may be comprehensively established at least for the cases described.
  • Thumbnail Image
    Publication
    Open Access
    Histoblot: A sensitive method to quantify the expression of proteins in normal and pathological conditions
    (Universidad de Murcia. Departamento de Biología Celular e Histología, 2023) Aguado, Carolina; Martín-Belmonte, Alejandro; Alfaro-Ruiz, Rocío; Martínez Moreno, Ana Esther; Luján, Rafael
    The histoblot (in situ immunoblotting) technique is a simple, reproducible, and sensitive method for protein detection that allows both protein quantitation and analysis of tissue distribution. This easy and fast method allows the direct transfer of native proteins from unfixed frozen tissue sections by mechanical pressure to an immobilizing matrix. Proteins are directly blotted onto nitrocellulose membranes that are then immunolabelled similar to a western blot, but the result is an immunohistochemical imprint of the section retaining all proteins. The histoblot combines advantages of western blot and immunohistochemical methods and yields optimal accessibility of proteins blotted on membranes whilst also preserving anatomical resolution. In addition, it avoids chemical modifications, crosslinking, or semi-denaturation of proteins, which can alter the access of antibody to epitopes, as introduced by conventional immunohistochemistry. Therefore, the histoblot often enables the use of antibodies that do not recognise the target protein in fixed tissue samples. This method has become a trusted alternative to reveal and compare the regional distribution and expression profile of different proteins in the brain in physiological and pathological conditions. In addition, the technique exhibits a high subregional resolution, although is not suitable to unravel protein distribution at the cellular and subcellular levels. In this review, we introduce the histoblot procedure used in our laboratory on brain sections for the identification of quantitative changes of neurotransmitter receptors, ion channels and other signalling molecules in the brain. We also discuss the potentialities, limitations, and fundamental principles of this technique.
  • Thumbnail Image
    Publication
    Restricted
    Longitudinal monitoring of anti-saliva antibodies as markers of repellent efficacy against Phlebotomus perniciosus and Phlebotomus papatasi in dogs
    (Wiley, 2019-11-18) Risueño Iranzo, José; Spitzová, T.; Bernal Gambín, Luis Jesús; Muñoz Hernández, Clara; López, M. C.; Thomas, M. C.; Volf, P.; Infante, J. J.; Berriatua Fernández de Larrea, Eduardo; Sin departamento asociado
    A 2-year longitudinal study of enzyme-linked immunosorbent assay (ELISA) antibodies against Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae) sandfly saliva was performed in 32 Beagle dogs treated preventively with an imidacloprid–permethrin topical insecticide in an endemic area in Spain. Dogs were grouped into three sandfly exposure groups according to the time of inclusion in the study. Assays analysed immunoglobulin G (IgG) against salivary gland homogenates (SGH) of both species and recombinant P. papatasi rSP32 and P. perniciosus rSP03B proteins in serum. The dogs were participating in a Leishmania infantum (Kinetoplastida: Trypanosomatidae) vaccine trial and were experimentally infected with the parasite in the second year. No dog acquired natural L. infantum infections during the first year, but most developed anti-saliva antibodies, and median log-transformed optical densities (LODs) were seasonal, mimicking those of local sandflies. This indicates that the repellent efficacy of the insecticide used is below 100%. Multi-level modelling of LODs revealed variability among dogs, autocorrelation and differences according to the salivary antigen and the dog's age. However, dog seroprevalence, estimated using pre-exposure LODs as cut-offs, was relatively low. This, and the fact that dogs did not become naturally infected with L. infantum, would support the efficacy and usefulness of this imidacloprid–permethrin topical insecticide in canine leishmaniasis control.
  • Thumbnail Image
    Publication
    Open Access
    Optimisation and validation of immunohistochemistry protocols for cancer research
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Ella-tongwiis, Peter; Makanga, Alexander; Shergill, Iqbal; Hughes, Stephen Fôn
    Background. Immunohistochemistry (IHC) has become a valuable laboratory technique for diagnosing, evaluating metastasis and informing treatment selection in several cancers. Standardization however remains a limiting factor in IHC. The main aim of this research study was to optimise, validate and standardize antibodies and IHC protocols for cancer research. Methods. Seven monoclonal mouse and rabbit antibodies were optimised using formalin-fixed paraffin embedded (FFPE) human tissue blocks. 4um sections of FFPE block were stained using the Roche Ventana XT or Ventana ULTRA IHC automated analysers. This study modified manufacturer recommended protocols by using a unique antigen retrieval method, adding an amplification step, varying primary antibody incubation times, as well as using the Roche Ventana Ultraview detection system. Results. Optimum antibody localisation was observed in modified IHC protocols in comparison with manufacturer recommended protocols for antiCEACAM-1, anti-CD31, anti-COX-2, anti-HER-2/neu, anti-S100P, anti-thrombomodulin and anti-VEGFR-3. Majority of antibodies required more than one modification of the initial protocol. For anti-VEGFR-3 optimum staining was observed following 4 protocol modifications. Conclusions. This study has optimised and standardized several tissue-based biomarkers that may be, in the future, used to screen, diagnose and monitor patients with certain cancer, such as bladder cancer. Accurate data on optimised protocols reduce time and resources wasted on experimental protocols, and ultimately help identify biomarkers or biomarker panels, which may be used to select treatment regimens for various cancers.
  • Thumbnail Image
    Publication
    Open Access
    Single-cell spatial proteomics
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2025) Liyanage, Senal; Guo, Jia; Biología Celular e Histología
    Recent advancements in single-cell spatial proteomics have revolutionized our ability to elucidate cellular signaling networks and their implications in health and disease. This review examines these cutting-edge technologies, focusing on mass spectrometry (MS) imaging and multiplexed immunofluorescence (mIF). Such approaches allow high-resolution protein profiling at the single-cell level, revealing intricate cellular heterogeneity, spatial organization, and protein functions within their native cellular contexts. MS imaging techniques offer unprecedented high-dimensional resolution and provide detailed insights into their subcellular protein localization and abundance. mIF enables rapid and high-throughput protein profiling, enhancing its accessibility for diverse research and clinical applications. This review assesses the current challenges associated with these methodologies and also discusses the potential solutions to overcome these obstacles. The integration of spatial proteomics with other systems biology approaches holds great promise for enhancing our understanding of complex biological systems. It could also lead to significant advancements in molecular diagnostics and personalized treatment strategies.