Immunoassay Labeling Techniques PDF

9/11/2022 Lecture No. 4 Chapter 12 Labeling Techniques in Immunoassay Gheyath K. Nasrallah, PhD Learning Objectives Compare heterogeneous and homogeneous immun...

9/11/2022 Lecture No. 4 Chapter 12 Labeling Techniques in Immunoassay Gheyath K. Nasrallah, PhD Learning Objectives Compare heterogeneous and homogeneous immunoassays. Name at least three types of labels that can be used in immunoassay. Describe chemiluminescence. Describe and compare chemiluminescence, enzyme immunoassay (EIA), and immunofluorescence techniques.  Briefly compare direct immunofluorescent, inhibition immunofluorescent, and indirect immunofluorescent assays  Student presentation: describe principle flwocytometry and immunophenotyping. 1  9/11/2022 Types Immunoassay the solidphase in heterogenous immonoassaycould be Heterogeneous immunoassay: the latex or magnetic beads using slides or other tools Solid phase: requires washing steps to remove unbound Ags or Abs can be competitive and noncompetitive. Homogeneous immunoassays: liquid phase with no washing (no separation) faster and easier to automate than heterogeneous immunoassay has competitive formats. Types of Immunoassay 2  9/11/2022 Radioimmunoassay (RIA) the most sensitive immunoassay  Radioactive isotopes such as radioactive Iodine isotopes (I125 or I131) or radio active Carbon produces gamma ray  Advantage: very sensitive (gold standard in some assays)  Disadvantage: 1- hazardous 2- short half life time of the kit 3- needs special equipment for measuring results (Gamma scintillation counters) P: protons N: neutrons 3  9/11/2022 Enzyme Immunoassay (EIA) Enzyme-linked immunosorbent assay (ELISA) Designed to detect Ags or Abs by producing an enzyme- triggered color change Use a nonisotopic label Enzyme Immunoassay 2 Ag detection: Direct detection, e.g. HBsAg detection of Ag A b Ab detection: indirect detection, e.g., anti-nuclear Ab (ANA)] 3detection of Ab A EIAs: three different type: b Noncompetitive EIAs: direct and indirect method Competitive EIAs Capture EIAs 4  9/11/2022 ELISA 1- Non-competitive (direct and indirect detection) 2- Competitive 3- Captured Vidio: http://www.piercenet.com/method/overview- elisa Figure 12-1 Principle of solid- phase EIA Direct sandwich ELISA we wash to remove excess Video: http://www.youtube.com/watch?v=q8-bBetS00Q the higher the intensity the higher the conc 5  9/11/2022 the first one should be polyclonal and the second will monoclonal Indirect ELISA Indirect sandwich Direct sandwich anti human we can label more antibodies so we are antibodies are amplifying the signal the secondary makes the assay cheaper ab Used for detection of Detection of Antigen by Sandwich assay antibodies Fc region specific We use anti-human antibodies (anti-IgG/anti-IgM)that are specific for IgG or IgM 2-competitive ELISA is a colormetric assay the highest conc of ab will bind faster best for quantitation because we now exactly how much we put of the serum horse radish peroxidase is the most common enzyme 6  9/11/2022 caot the plate with anti IgM or anti IgG ( dpending on the antibodies) to caputre the antibody to adsorp 3- Captured EIA  designed to detect a specific type of Ab, such as IgM or IgG against specific Ag such as CMV- IgM, rubella-IgM, or Toxoplasma IgM.  Ab specific for IgM or IgG is attached to a solid-phase surface, the patient specimen potentially containing IgM or IgG is added. Specific antigen is then added. used in research more than diagnosis Enzymes used as labels most common most stable Horseradish peroxidase (PER or HPR) peroxidase it converts H2O2 to NO2 Alkaline phosphatase Glucose-6-phosphate dehydrogenase Beta-galactosidase 7  9/11/2022 Enzyme Immunoassay Enzymes used as labels must have: A high amount of stability What are the characterstis? A/B/C/D/all of the above Extreme specificity does not have epitopes very similar to the ab or ag that I am looking for Absence from the antigen or antibody No alteration by inhibitor with the system if we are examining samples like stool or sputum Signal amplification streptavidin-bioten can conjugate up to 100 enzymes mainly used for quantification the ab is called bionaltyed ab Add: Patient sample, then wash Ab-Biotin (B), then wash Streptavidin (STR) -Bioten-Enzyme (PER), then Add substrate: tetramethylbenzidine (TMB) Stop the reaction with acid 8  9/11/2022 Signal amplification biotinyl-tyramide: amplifying reagent can bind to HRP and any protein in the well. http://www.ebioscience.com/kno wledge-center/product- TMB: Tetramethylbenzidine line/elisa/high-sensitivity-elisa- B: Biotin kits.htm STR: Streptavidine HRP: ? Hypersensitive response and pathogenicity Chemiluminescence Used extensively in automated immunoassays Chemiluminescence refers to light emission produced during a chemical reaction Two formats: competitive and sandwich immunoassays. we have excitation and …. in elisa only absorbance Video: https://www.alpco.com/a-comparison-of- colorimetric-and-chemiluminescence-elisas 9  9/11/2022 Chemiluminescence [A] + [B] + [◊] → [Products]+ light (protons: counted )  [A] is luminol (C8H7N3O2) chemiluminescnece material  [B] is hydrogen peroxide (H2O2): oxidizing agent  [◊] is catalyst: e.g peroxidase (HRB)  [Products]: 3-aminophthalate (3-APA) emit florescent blue light at 425 Photons or fluorescent light luminol + H2O2 + HRP → 3-APA Text (-APA emit fluorescent light at 425 nm) Reagent 1: H2O2 + HRP (conjogated to Ab) → O2- + H2O Reagent 2: O2- + Luminol → 3-APA [emit light (photons) at 425 nm]: counted by photon counter (fluorescent filter). Excitation wave length: 425 produces the light Emission wavelength: 489 Common also in Western blot. Chemiluminescence nd Eectro Chemiluminescence ECL in rosch they can detect electrochmiluminescence [A] + [B] → [◊] → [Products] + light (emission at 425): photons [A] could be: 1- luminol 2-acridinium esters : need detergent as catalyst substrates 3-peroxyoxalates 4-dioxetanes Electrode 5-tris(2,2′bipyridyl)-ruthenium or ruthenium and tripropylamine (TPA) complex [Ru(bpy)3+ ] : generate 10  9/11/2022 Sandwich Chemiluminescence Immunoassay here beads are the solid phase (Redrawn from Jandreski MA: Chemiluminescence technology in immunoassays, Lab Med 29:557, 1998.) it could be magnet based beads or latex but if we are going to use latex we need to centrifuge to remove the latex Competitive Chemiluminescence Immunoassay quantitation 11  9/11/2022 Enzymes are typically used for indirect labels.  Indirect labels are attached to antibodies, antigens, and DNA probes, we label our Ag but it needs to be stable and big enough to conjugate with the enzyme Enzyme labels often used Alkaline phosphatase (ALP) Horseradish peroxidase (HRP) Beta-galactosidase (β-galactosidase) Recombinant apoaequorin (from the bioluminescent jellyfish, Aequoria). It is activated by reaction with coelenterazine (Reagent 1). Light emission at 469 nm is triggered by reaction with calcium chloride (Reagent 2). Automation & Chemilumenscent  Immulite 2000 : One of many clinical applications of chemiluminescence is a third- generation serum IgE (sIgE) method (ImmunoLite 2000, Diagnostic Products) that is a solid-phase (bead), two-step chemiluminescent EIA.  Allergens are covalently lined to a soluble polymer–ligand matrix, allowing immunochemical reactions to occur in liquid phases for random-access automation.  Other methods in automation: magnetic beads as the solid phase. 12  9/11/2022 Immunofluorescent Techniques Immunofluorescent Techniques they are mainly used in autoimmune disease Direct Immunofluorescent Assay (DFA) Inhibition Immunofluorescent Assay Indirect Immunofluorescent Assay (IFA) Labeling Fluorescent: FITC: fluorescein isothiocyanate cheap and stable Non-Fluorescent: HRP, ALP, and avidin-biotin conjugated enzyme: These reagents have the advantage of requiring only a standard light microscope. 13  9/11/2022 Direct Immunofluorescent Assay 1. Direct: detecting 1. In direct: detecting Ab in the Ag in the tissue tissue (ANA) lupus disadvantage:need expertise and flourescent microscope anti nuclear antibodies - to detect use the anti human antibodies conjugated with flourscence readily preped by company sometimes we will get unspecific binding fix tissue -> s=cover it with Anti(antigen) antibodies that this conjugated Indirect Immunofluorescent Assay (IFA) Autoimmune disease Most important ANA 14  9/11/2022 Antinuclear Antibody (ANA). Homogeneous or Diffused: A solid staining of the nucleus with or without apparent masking of the nucleoli. Nuclear antigens present: dsDNA, nDNA, DNP histone. Disease association: High titers are suggestive of systemic lupus erythematosus (SLE); lower titers are suggestive of SLE or other connective tissue diseases. Antinuclear Antibody (ANA). Nucleolar: Large, coarse, speckled staining within the nucleus, generally less than 6 in number per cell, with or without occasional fine speckles. Nuclear antigens present: 4-6S RNA and other unknown nuclear antigens. Disease association: High titers are prevalent in scleroderma and Sjogren's syndrome 15  9/11/2022 Antinuclear Antibody (ANA). ?: The chromosome region of mitotic cells demonstrates the same smooth, homogeneous staining pattern Antinuclear Antibody (ANA). Centromere: A discrete, speckled staining pattern. The nuclear speckles are very discrete and usually in some multiple of 46 (23-46 speckles per nucleus). Nuclear antigens present: Chromosomal centromere (kinetochore). Disease association: Highly suggestive of CREST syndrome, a variant of progressive systemic sclerosis (PSS). 16  9/11/2022 Inhibition (competitive Immunofluorescent Assay A blocking test in which an antigen is first exposed to unlabeled antibody, then to labeled antibody, and is finally washed and examined. Emerging Labeling Technologies digoxin is a substrate /material that is bound here in cyto sito? hyberdization http://commons.wikimedia.org/wi ki/File:FISH_(Fluorescent_In_Situ_ Hybridization).jpg the difference between chemiluminsecne and flourcense there is because not us e enzyme lly we do ce ( usua zy m e or flourscen no en could be 17  9/11/2022 Emerging Labeling Technologies  FISH: Fluorescent in situ NBT/BCIP hybridization: or Fast Red  RNA detection: gene Alkaline Phosphatase expression can be used for identifying cells producing IL- Antibody 4 of IL-1, etc. Purple stain or  RNA probe is labeled with Red stain DIG or FITC labeled probe DIG or directly labeled with Target Fluorescent molecule nucleic acid  DNA detection:  Tyramide signal amplification (TSA): Summary LABELED REACTIONS 1. Definition: Antibodies labeled with a fluorescent dye are used to detect an antibody or antigen.  Methods a. Direct immunofluorescence: Conjugated (ftuorescent labeled) reagent antibody reacts with an antigen in a clinical sample to form an antigen-antibody complex. b. Indirect immunofluorescent assays: Antigen reacts with unlabeled antibody forming an antigen-antibody complex that is then complexed with a labeled antihuman antibody, creating an antibody-antigen-antibody "sandwich.“ c. Biotin-avidin immunofluorescence: This is an indirect assay in which the detection system is modified by using a biotin-labeled antibody followed by avidin-labeled ftuorochrorne. This extra step increases the specificity and sensitivity of the assay.  Commonly used fluorochromes: include fluorescein isothiocyanate (FITC), R- phycoerythrin, quantum red, tetramethyl-rhodamine isothiocyanare, Texas red, phycocyanin, acridine orange, and propidium iodide. 18  9/11/2022 Example of Indirect immunofluorescent: Antinuclear antibodies (ANAs): Antibodies to nuclear antigens are present in many systemic autoimmune diseases, such as systemic lupus erythematosus, mixed connective tissue disease, and rheumatoid arthritis. This rest is used for diagnosing, developing a prognosis, and monitoring treatment of certain autoimmune diseases. a. Indirect immunofluorescence is used for ANA screening. b. Procedure: Cultured cells on a microscope slide are incubated with patient serum. The cells are washed, then incubated with antihuman immunoglobulin conjugated with fluorescein. The slide is washed again, then viewed using a fluorescent microscope. Enzyme-Linked lmmunosorbant Assays (ELISAs) I. Enzyme-labeled reagents are used lo detect antigens or antibodies. 2. Enzyme must be stable, specific, and cannot bind to antigen or antibody independently. 3. A colorless substrate is metabolized by the enzyme into a colored compound. The intensity of the color is directly proportional to the amount of enzyme present. Cellular Assay: presented by Roan (flowcytometrey) A. Lymphocyte Subsets I. T cell subsets a. Enumeration of T cells is important in assessing immune response. b. Monoclonal antibodies are used in conjunction with flow cytometry to identify cell markers such as CDI, CD2, CD3, and CD4. 2. B cell subsets a. Classical test: Labeled antibody to surface membrane imrnunoglobulin b. Monoclonal antibodies are now used in conjunction with flow cytometry to identify CD 19 or CD20. 3. Lymphocyte phenotyping in human immunodeficiency virus (HIV) infection a. HIV kills T helper cells, and the primary viral receptor for infection is CD4. b. CD4 and CDS markers are monitored during treatment. If the CD4 count falls below 200/µL, the patient is susceptible to opportunistic infections. 4. Other cells identified by flow cytornetry and monoclonal antibodies 19