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Principles of immunological Techniques MLU 3248 by H Weerawarna Visiting Academic Open University of Sri Lanka Immunoassay Relative concentrations of different substances in blood. 10 mmol/L 100 10 m mol/L 100 10 100 pmol/L Glucose Calcium Mg Sodium creatinine Iron, Immunoglobulin Oestriol T4 Progesteron T3 Insulin TSH Hormones Drugs proteins Use of immunoassay tumour markers Vitamins pathogens Immunoassays Immunological reactions Sensitive Antigen + antibody + detectors Highly specific RIA FIA EIA Radio active ELISA Fluorescence Enzymes Immunodiagnostic techniques Range of techniques directly visible Agglutination Precipitation Immuno-Immuno fluorescence Manual RIA ELFA ELISA Automated Antigen As a result of the reaction of the body to immunogen antibodies are produced Antibody + antigen antibody antigen complex Principle of agglutination - Antigen detection ? Specific antibodies are linked to a solid phase solid phase latex red blood cells agglutination Latex agglutination Positive Negative latex agglutination Principle The Direct Pregnancy test is based upon the latex agglutination reaction between latex particles coated with anti-hCG antibodies and hCG present in the test specimen. • The presence of hCG in the urinary specimen results in an agglutination • which is visually differentiated from the non-agglutinating negative control. Characteristics of precipitation reactions Figure 17.6 Precipitation curve Passive Immunodiffusion Passive diffusion method in which a concentration gradient is established for an antigen and /or antibody. • Diffusion reactions without electric current to speed up reaction. • Rate of Diffusion depends on – Size of particles – Temperature – Gel viscosity – Amount of hydration – Interaction between matrix and reactant. Immunodiffusion Techniques Radial Immunodiffusion a single diffusion technique where Ab is put into gel and Ag is measured by the size of a precipitin ring formed when it diffused out in all directions from a well cut into the gel. Ouchterlony Double Diffusion Both Ab and Ag diffuse from wells into a gel medium Medium • Agar – high molecular weight complex polysaccharide from seaweed. Agar has strong –ve charges. • Agarose - purified agar , no charge, interaction between gel and reactants are minimized. • Used to help stabilize the diffusion provess and allow visualization of precipitin bands. Radial Immunodiffusion (RID) • Antigen from the sample is added to antibody rich media. • Antigen and antibody continue to react until the zone of equivalence is reached. • The area of ring is a measure of the antigen concentration. Measurement of diameter of precipitation zone Micrometer eye piece Ouchterlony Double Diffusion Fusion of lines at their junction to form an arc serologic identity / presence of common epitope. Crossed lines demonstrates 2 separate reactions compared antigens shard no common epitopes Fusion of 2 lines with spur partial identity Rocket electrophoresis Enzyme Linked Immunosorbent Assay (ELISA) Introduction The enzyme linked immunosorbent assay (ELISA) is a versatile method for detecting and quantifying a specific protein in a complex mixture. Originally described by Engvall and Perlmann (1971), the method enables analysis of protein samples. immobilized in microplate wells using specific antibodies. The technique has revolutionized immunology and is commonly used in medical research laboratories. ELISA also has commercial applications, including the detection of disease markers and allergens in the diagnostic and food industries. Basic elements in ELISA All the variants of ELISA have same basic elements: 1. Coating/Capture: direct or indirect immobilization of antigens to the surface of polystyrene micro plate wells. 2 Plate Blocking: addition of irrelevant protein or other cover all unsaturated surface-binding sites of the micro plate wells. 3. Probing/Detection: incubation with antigen-specific antibodies that affinity-bind to the antigens. 4. Signal Measurement: detection of the signal generated via the direct or secondary tag on the specific antibody. molecule to Enzyme-linked immunosorbent assay An antibody sandwich ELISA Sandwich method Ab WASHING + STEP Conjugate Solid Sample phase E E WASHING E STEP Competitive binding Immunoassay, EIA Colour reaction in ELISA 3,3'-5,5' tetramethylbenzidine (TMB) TMB One-Step Substrate System is a stabilized, ready-to-use chromogenic substrate solution for ELISA when peroxidase is the enzyme label. TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidse. 3,3'-5,5' tetramethylbenzidine (TMB) The TMB One-Step Substrate System contains 3,3'-5,5' tetramethylbenzidine (TMB) and hydrogen peroxide in an organic solvent/buffer solution. Upon oxidation, TMB forms a blue reaction product that can be measured at 605 nm. Upon acidification (to stop the enzymatic reaction) the reaction product turns yellow with an absorbance peak at 450 nm. Sandwich ELISA protocol 1. Coat primary antibody onto microplate. 1a. Allow antibody adsorption and block unoccupied sites with neutral protein (BSA). 2. Add antigen sample to be detected into each well. Incubate 30 min at 370 C. 3. Add second primary antibody against antigen and HRP-conjugated secondary antibody (antibody mix) into each well. Incubate 30 min at 370 C. 4. Develop colorimetric reaction with appropriate substrate. Incubate 15 min at room temperature. 5. Stop reaction with 3M H2SO4. Read absorbance in ELISA spectrophotometer and quantitate relative antigen levels. ELISA instrumentation Microplate washer multichannel pipette. ELISA plate, 96 wells Microplate reader MICROPLATE PHOTOMETER Microplate Photometer components MICROPLATE PHOTOMETER Microplate Photometer components Enzyme Multiplied Immunoassay Techniques (EMIT) EMIT has a wide application in therapeutic and illicit drug monitoring. In this type of assay, a sample of interest with the analyte (drug) is added to a fixed quantity of enzyme – bound drug and the anti – drug antibody. After the addition of substrate, absorbance measurement s are taken at time intervals to determine the speed of the enzyme reaction. Assay components drug antibody substrate enzyme bound to drug Enzyme Multiplied Immunoassay • Techniques have a wide application in therapeutic and illicit drug monitoring. In this type of assay, a sample of interest with the analyte (drug) is added to a fixed quantity of enzyme – bound drug and the anti – drug antibody. After the addition of substrate, absorbance measurement s are taken at time intervals to determine the speed of the enzyme reaction. Enzyme Multiplied Immunoassay Techniques (EMIT) The more free drug in the sample, the faster the enzyme reaction because only the unbound enzyme-drug complexes are capable of binding the substrate. The method can be used for whole blood, serum or urine. Enzyme Multiplied Immunoassay Techniques (EMIT) Ag being measured competes for the Ab binding sites with the antigen that has been labeled with an enzyme. The antibody reagent blocks any enzymatic activity when it is bound with the reagent enzyme Ag complex , preventing the formation of the product with a substrate. The free Ag –enzyme complexes compete with the Ag (in the sample), forming a colour chanege which are proportional to the concentration of the Ag present in the tested sample Enzyme Multiplied Immunoassay Techniques (EMIT) Procedure - mix sample containing drug with antibody. Incubate add fixed quantity of enzyme bound drug. Incubate. add substrate and cofactor. incubate. Measure absorbance 15-45 seconds after substrate addition. - Absorbance -> reaction rate ->drug concentration. - nonlinear relationship between absorbance and concentration. Enzyme Multiplied Immunoassay Techniques (EMIT) • Use for drug, hormone and metabolite determination. • EMIT is widely used in therapeutic and illicit drug monitoring dg: serum Digoxin, Cocaine, marijuana Advantages of EMIT homogeneous, there is no need of washing excess of labelled enzymes. Long shelf-life assays are well established. More specialty assays are available it is a competitive assay. Reagent separation is not required . Faster smaller molecules are measured Disadvantages of EMIT many interferences less sensitive compared to ELISA in hormone determination false negatives are common tolmetin and aspirin metabolites Radioimmunoassay (RIA) Radioimmunoassay (RIA) is laboratory technique for measuring the levels of substances which have low concentrations in the blood. Eg hormones. Sensitive and detection limit 10 -15 mol/l 10 3 -10 6 times more sensitive than colorimetric assay HISTORY OF RIA Principle of RIA 1. plasma sample from the patient containing the substance whose concentration is to be measured. 2. The same substance with a radioactive label, eg use of chloramine-T for labeling proteins with high activity of radioiodine. 3. Receptors which bind only the substance of interest - antibody Production of antisera To produce the antiserum, the antigen is injected subcutaneously or intraperitonelly into and experimental animal, usually the rabbit, together with an adjuvant to stimulate the immune system. Large molecules with MW over 10000 are naturally antigenic and cause the production of antibodies. Small molecules such as thyroid and steroid hormones and drugs are not antigenic, made antigenic by process called haptenisation, molecule is linked to a large molecule as albumin. After booster dose animal is bled to produce antiserum (polyclonal) Radioactive label The choice of a radio active label for the antigen will depend on its specific activity • Physical half life • Biological and chemical stability 3H, 51Cr, 57Co, 59Fe, 125I 125I , ½ life 60 days Iodine-125 Radioiodination Iodination of peptides and proteins, as well as small molecules, results in materials with high specific activity. Generally, Iodine-125 is incorporated into available tyrosine residues under oxidative conditions. Various compatible oxidative reagents of differing strengths exist (e.g. Chloramine-T, Iodogen, Lactoperoxidase) and can be chosen to accommodate sensitive molecules. Additionally, for compounds lacking a tyrosine or histidine or are simply incompatible with other iodination techniques, labeling of free amines can occur through the use of [125I]Bolton-Hunter Reagent. Due to the wide variety of compounds amenable to 125I labeling and a number of variables involved to achieve desired specifications, please contact us to discuss your project’s needs. Iodination of tyrosine - chloramine-T method The principle of RIA The principle of RIA (cont.) If total Ab input is kept constant, the value of B/F is a measure for the total Ag input Measurement of Bound/Free Tracer The Standard Curve To construct a binding inhibition curve based on known ( standard) amounts of antigen, for use in interpolation of unknown samples. Counting techniques The counting of the activity of the samples done by using – gamma counter or g emitting labels eg; 125 I – Liquid scintillation counter for 3 H labeled assays. Multiwell gamma counters capable of data processing, plotting standard curve and result calculation are use for efficient counting. scintillation detector configurations Photomultiplier tube (PMT) • Photomultiplier tube. Its basic structure is an evacuated cylinder enclosed in glass, with a photocathode on one end, an anode at the • opposite end, and small curved dynodes in between. The electrical potential to the dynodes is what causes multiplication of the electrical signal • created at the photocathode. The passage of an electron through the focusing grid, its interaction with the first dynode, and its multiplication at the second dynode is shown, using a multiplication factor of 3. Photomultiplier tube. Block diagram of a scintillation detector. The detector/PMT assembly is usually separated from the electronics module by a cable e Multi well gamma counter Standard curve - RIA VIDAS PRINCIPLE Courtesy of BioMerieux, France VITEK IMMUNO DIAGNOSTIC ASSAY SYSTEM VIDAS An innovative test format Ready-to Readyto--use reagents in the 1010-well strip Specific antibodies coated onto the SPR SANDWICH Detection of antigens 4-methyl umbelliferyl phosphate Bound Ab Alkaline phosphatase-labeled Ab Ag to be detected COMPETITION Detection of haptens 4-methyl umbelliferyl phosphate Bound Ab Alkaline phosphatase- labeled hapten Hapten to be detected INDIRECT ELISA Serology 4-methyl umbelliferyl phosphate Ab to be detected Alkaline phosphatase- labeled Ab Bound Ag IMMUNOCAPTURE Detection of specific IgM 4-methyl umbelliferyl phosphate Ab to be detected alkaline phosphatase-labeled Ab Anti-IgM Ag in liquid medium DYNAMICAL REACTIONS EXAMPLE SPR Strip Washing buffer Conjugate Sample Diluent (Prewash) Substrate photodiode EXAMPLE OF PROCEDURE Detection of antigens Cycle in and out SPR E E E E E E Target organism E SAMPLE WASHING BUFFER CONJUGATE IMMUNOLOGICAL REACTION SUBSTRATE ENZYMATIC REACTION - 7-1 READ FLUORESCENCE EXCITATION EMISSION Filters Lens Xenon Flash Lamp Photodiode - 7-2 VIDAS : adapted to lab requirements As easy as 1, 2, 3... 1- Load sample 2- Insert test 3- Press button series : 1 to 30 tests / run 1 or more pathogens / run Rapid (Results in 2424-48 hours) Software Standardization Controls Traceability Walk Away system VIDAS : adapted to lab requirements High accuracy sensitivity, specificity > 98% Bar-code BarStandardization and traceability Ready-toReadyto-use reagents No preparation step Optimization of reagents Long shelf life Cost reduction Reduced lab work Reduced time Reduced lab materials