Transcript elisa
Analytical and preparative methods based on primary antigen-antibody binding Immunoaffinity chromatography, ELISA, immunoblot, immunohistochemistry Affinity purification of antibodies using an antigen-sorbent column column polymer beads Column content Covalently bound antigen Addition of antibodies to be purified Binding Washing elution Antigen-specific polyclonal antibodies The reverse setup is also used Purification of antigen on antibody-immunosorbent column polymer beads column Loading the antigen mixture Binding Wash Column content Elution Covalently bound antibodies ELISA Enzyme Linked Immune Sorbent Assay ELISA plate Well enzyme linked immune sorbent enzyme Antibody conjugated with enzyme Antigen/antibody adsorbed to solid surface Enzyme activity in ELISA is directly proportional to the amount of antigen present Enzyme activity is measured by the color reaction due to conversion of substrate Similar principle applies to many other antibody-based detection methods Basic setups in ELISA, immunohistochemistry, flow cytometry Indirect method Direct method Label Label Secondary antibodies Primary antibodies Antigen Basic setups in ELISA, immunohistochemistry, flow cytometry Enzyme/anti-enzyme system PAP – peroxidase / anti-peroxidase APAAP – alkaline phosphatase / anti- alkaline phosphatase Enzyme Enzyme-specific antibody, Same isotype as the primary antibody Primary antibody Antigen Secondary antibody Basic setups in ELISA, immunohistochemistry, flow cytometry Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity ) Basic Avidin-enzyme complexes ABC Avidin-biotin enzyme complexes Biotin-enzyme complex Avidin Biotinylated antibody Antigen Examples of direct, indirect and competitive ELISA systems Steps of the basic indirect ELISA Detection of antigen or specific antibody Adsorption of antigen (coating) Removal of excess antibody Saturation of uncovered surface area with protein such as BSA, casein etc.. Addition of Agspecific antibodies Addition of Secondary Ab conjugated with enzyme Addition of chromogenic substrate Removal of excess antigen Removal of excess protein Steps of the combined sandwich ELISA For antigens present at low concentration in complex biological samples Coating with Agspecific „capture” antibody Blocking free plastic surface with inert protein Addition of antibodycontaining sol. Addition of biotinylated antibody specific to a different epitope on target protein Addition of avidinconjugated enzyme Addition of substrate Removal of excess enzyme Removal of unbound material Removal of unbound protein Removal of unbound material Competitive ELISA Highly sensitive method used to detect and quantitate small amounts of antigen in complex biological samples Antigen in solution and on the solid surface compete for the binding site of labeled specific antibody. - Coat with antigen, blocking - Add experimental sample that contains or lacks antigen -Add labeled antibody, --- Binding - Washes - Add substrate ELISA Background due to non-specific binding to plastic Labeled antibody bound to plastic directly or indirectly gives the same signal as those that bind specifically Nonspecific binding can be controlled by: •Saturation of free plastic surfaces •Using detergents ELISA plates - results The chromogens used for detection are CARCINOGENOUS! Characterization of antigens by Western blotting Western Blot • The use of antibodies in molecular biology is widespread • It is probably most often encountered in Western analysis • SDS-PAGE gel resolved into single protein bands (overlap possible) • Presence of a protein is determined by hybridizing the proteins, transferred or applied to a membrane, with the relevant antibody Protein Standard sample Antibody recognizes epitope in specific protein SDS-PAGE Membrane Western blot Western Blot • Used to detect specific proteins in a sample • Proteins separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDSPAGE), transferred to a membrane • Primary (1st) antibody (monoclonal or polyclonal) used to detect protein • Enzyme linked 2nd antibody (e.g. horseradish peroxidase-linked) used to detect 1st antibody Enhanced chemiluminescence (ECL) Immobilized proteins Primary Fabantibody Fc •In the presence of H2O2, horseradish peroxidase (HRP) oxidizes diacylhydrazides such as luminol • Directly after oxidation, luminol is in an exited state, and emits a photon to return to the ground state • This photon can be detected with a film or a camera • Light emission can be enhanced by ~1000-fold with phenolic compounds such as 6-hydroxybenzothiazole (enhancer) Horseradish peroxidase H2O2 Epitope on protein surface Secondary antibody Membrane H2O luminol luminol h enhancer Detection Immunohistochemistry Labeled antibodies added to fixed tissue sections detect the distribution of the chosen antigen within the tissue or within the cells of a particular tissue • Immunfluorescence •Fluorescent dye coupled to antibody FITC – fluorescein isothiocyanate PE – phycoerythrin • Immunoenzyme method • enzyme-coupled antibody P – peroxidase PA – alkaline phosphatase (Substrates converted into an insoluble compound) Immunohistochemistry Fixation Tissue sample Freezing Sectioning Section before staining Immunohistochemistry ABC Method Enzim X Avidin Biotin Secondary antibody Primary antibody Slide Cells Tissue sample Immunohistochemistry Acute bronchopneumonia (hematoxilin- eozin staining) Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma) Immunohistochemistry using fluorescent detection Anti-nuclear autoantibodies in SLE (FITC) Immunohistochemistry using fluorescent detection (TRITC) Detection of actin microfilaments A fixed and permeabilized skin fibroblast. Mitochondria were labeled with mouse IgG (anti–OxPhos Complex V) and visualized using goat anti–mouse IgG conjugated with orange-fluorescent Alexa Fluor 555. F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin (a mushroom toxin), and the nucleus was stained with TO-PRO-3 iodide Peroxisome labeling in fixed and permeabilized pulmonary artery endothelial cell. Peroxisomes were labeled using an antibody directed at peroxisomal membrane protein 70 and detected with Alexa Fluor 488–labeled goat anti–mouse IgG. Mitochondria were stained with MitoTracker Red prior to fixation; nuclei were stained with blue-fluorescent DAPI. Acquired Immune Deficiency Syndrome – AIDS Certain infectious microorganisms can supress or subvert the immune system. At the beginning of this century, when tuberculosis was the leading cause of death and fully half the population was tuberculin-positive, it was well-known that an intercurrent measles infection would cause a well-contained tuberculosis infection to run rampant and result in death. The mechanism responsible is now known to be the supression of IL-2 synthesis after binding of measles virus to CD46 on macrophages. Some of the microorganisms that supress immunity act by infecting lymphocytes. The human immunodeficiency virus (HIV) presents a chilling example of the consequences of infection and destruction of immune cells by a microorganism. The T-cell surface CD4 molecule acts as a receptor for HIV. CD4 is also expressed on the surface of cells of the macrophage lineage and they too can be infected by this virus. The clinical latency is long, usually it means several years. During this period, the level of CD4+ cells and virus particles in the blood rapidly changes. When the rate at which CD4+ cells are being destroyed exceeds the capacity of the host to replenish them, their number decreases to a point where cell-mediated immunity falters. The failure of cell-mediated immunity renders to the host susceptible to fatal opportunistic infections. Case study: The Pinkerton-family: infected blood caused tragedy Benjamin Pinkerton was a US-navy lieutenant who saw service at Japan. He married with a japan woman during his service, who gave birth two healthy girls in 1987. She bore a boy four years later, who seemed healthy, as well. The boy got the routine DPT-vaccination and an oral polio-virus immunization. These vaccinations had no side-effect and the boy grew normally. At the age of six months he fell sick and started to lose weight. He had severe, chronic diarrhea with fever. Besides a chronic oral candidiasis, the boy got two otitis, one after the other . The navy doctors examined the baby several times and prescribed antibiotic but it proved ineffectual. Results of somatic examination: - body-temperature 38oC; - candidiasis on the lateral sides of tongue and on the mucosal surface of the oral cavity; - „diaper-pimples”, which is also caused by Candida infection; - at respiration a subtle, slurping noise was heard in each pulmonary lobes; Laboratory: - normal amount of leukocytes (6500/ml); - normal rate of leukocytes (neutrophyl 62%; lymphocyte 30%; monocyte 5%; eosinophyl 2%; basophyl 1%); - normal serum immunoglobulin levels: - serum IgG: 997 mg/dl (phys.: 800-1000 mg/dl); - serum IgM: 73 mg/dl (phys.: 50-150 mg/dl); - IgA: 187 mg/dl (phys.: 150-300 mg/dl); - normal amount of CD8 + T-cells, but the rate of CD4+ T-cells is very low, only 85/ml (phys.: 1000-1200/ml); - intradermal Candida-antigen did not evoke late-type hypersensitvity reaction; - results of ELISA and Western-blot analysis: HIV-antibodies in the serum; METHOD ELISA Western blot EXAMINED PROTEINS SAMPLE DETECTION Anti-HIV IgG and IgM antibodies serum (sometimes saliva or urine) with secondary (conjugate-) anti-IgG antibodies HIV capsid-proteins: p17, p24 , p6, p7 Envelop: gp41, gp120, gp160; serum with secondary (conjugate-) anti-IgG antibodies During HIV-diagnostics samples are always analyzed firstly by ELISA-method. In case of reactivity (serum positive) two more measurements are needed (2nd and 3rd analysis). If the 2nd and 3rd measurements show reactivity as well, the subject’s sample must be verificated: usually a Western blot or another ELISA is performed. After this finding they verified the parents: Both of them were HIV-positive. While Pinkerton was healthy, his wife was feeling unwell and complained about the swelling of her cervical lymphatic nodes. It turned out, that she was pregnant right before the boy’s birth. At the end of pregnancy the fetus had died and had to be removed by caesarean section. The operation was going well but – because of the loss of blood – she needed blood-transfusion (she got two units of blood). The baby got two severe infections in turn: Pneumocystis carinii- and Pseudomonas aeruginosa. He had serious cough with bloody spit (hemoptysis). A week after this he died. The parents got AZT- (zidovudin) therapy. While his wife died soon in respiratory failure, the lieutenant – in spite of his high serum HIV-antibody level – has not had symptoms yet.