1	CLSC320 Principles of Immunology Diagnostic Laboratory Immunology Program for Clinical Laboratory Science Unit - 13 Labeled Immunoassays
2	Unit – 13 Guidelines Reading assignment: Pages 270 - 289 of textbook Learning objectives: Those listed on page 271 of textbook Key terms: Those listed on pages 271 - 272 of textbook
3	Labeled Immunoassays - Principles Labeled antigens or labeled antibodies are used to detect the formation of antigen-antibody complexes
4	Labeled Immunoassays - Applications High specificity and sensitivity make these assays the methods of choice in detecting low quantities of antigens or antibodies
5	Labeled Immunoassays – Labels Used Radioisotopes e Enzymes e Fluorochromes e Chemiluminescences
6	Characteristics of Monoclonal Abs High specificity e Low cross-reactivity e Ease of labeling e High affinity e Stable
7	Radioisotopic Labels Types of radiation: beta ($) gamma (() Half-life: Definition: the time during which half of a substance will be converted to a product or eliminated by a process. in the case of radioactive half-life it is when half of the radioisotope has decayed (one nuclide transforms into another nuclide by emission of particles) Examples: ⁴²K ¹³¹I ¹²⁵I ⁵⁷Co ³H ¹⁴C 12.4 hours 8.1 days 60 days 270 days 12.26 years 5,730 years ³H (tritium), ⁴²K (potassium), ¹³¹I (iodine), ¹⁴C (carbon) ⁵⁷Co (cobalt), ¹²⁵I (iodine) ¹²⁵I (iodine) emits very low energy photons - scintillation detector
8	Enzyme Labels Definition: enzymes are highly specific proteins that alter the rate at which a reaction proceeds Examples: peroxidase (horseradish) alkaline phosphatase Characteristics of a good enzyme for clinical use: 9foreign - not present in patient=s body fluids being tested 9specific activity - not altred by attachment to ag or ab 9stable - activity does not change during course of assay 9cheap 9readily available 9long shelf-life 9produces colored products 9assay can be automated
9	Fluorochrome Labels Definition: a compound that has the property of absorbing light of one wavelength and emitting light of another wavelength Examples: 9fluorescein isothiocyanate 9phycobiliproteins green light red light Characteristics of a good fluorophore for clinical use: 9must not interfere with antigen-antibody binding 9must maintain their stability 9must be able to absorb excitation light in available wavelengths 9must be able to emit light in an appropriate wavelength
10	Chemiluminescence Labels Definition: a compound that when excited by a chemical reaction will emit light Major groups of chemiluminescence labels: 9acridinium esters blue light 9luminol derivatives blue light 9oxalate derivatives 9ruthenium complex 9dioxitane derivatives Clinical applications: 9direct labels 9indirect labels attached to Ag, ab, or DNA probe serve as substrates for enzymes
11	Types of Protocols Competitive Binding e Non-Competitive Binding
12	Competitive Binding Protocol - 1 Definition: a procedure in which labeled and unlabeled molecules (antigens or antibodies) compete for a limited number of specific binding-sites on binder or receptor molecules. Procedure: 9a commercially prepared label antigen is added in excess of available antigen-binding sites on the antibody molecule 9patient serum containing the unlabeled antigen is then added and will compete with the labeled antigen for antigen- binding sites on the antibody molecule 9a separation step is necessary to remove the unbound antigen (both labeled and unlabeled) from the test system 9an appropriate measuring device then determines the amount of labeled antigen left in the system (bound to ab)
13	Competitive Binding Protocol - 2 Procedural information: continued 9if the presence of antibodies in patient samples is to be measured then a commercially prepared antigen is adsorbed unto a solid phase and labelled antibody and patient sample are added and compete for epitopes on antigen 9in the reaction phase equilibrium must be achieved (all ag-ab complexes have formed) 9in the separation step it is necessary to have the ag-ab complexes formed bound to a solid surface so they are not washed out of the system
14	Soluble Phase – For Antibody
15	Soluble Phase – For Antigen
16	Solid Phase – For Antibody
17	Solid Phase – For Antigen
18	OSF - Hashimoto=s Thyroiditis On page 276AOne Step Further@ presents a more in-depth discussion of Hashimoto=s thyroiditis disease and it=s detection. This presentation is contained on a separate slide presentation called A One Step Further #14" The student may call up the slide program OSF-14 later or click on the arrow below to view slides now.
19	Non-Competitive Binding Protocol - 1 Definition: a procedure in which a reagent antigen is adsorbed unto a solid phase and the patient=s sample is added (antibody). An labeled anti-human globulin (AHG) is then added and the amount of bound, labeled AHG is then measured. Procedure: 9a commercially prepared antigen is adsorbed unto a solid phase 9patient serum containing the unlabeled antibody is then added and will bind with the labeled antigen forming Ag-Ab complexes bound to solid phase 9a separation step is necessary to remove the unbound immunoglobulins from system
20	Non-Competitive Binding - 2 Procedure: cont=d 9a commercially prepared labeled antihuman globulin is then added which will bind to the antibody complexed to the bound antigen 9another seperation step is required to remove any unbound labeled AHG from the system
21	Solid Phase – For Antigen
22	Solid Phase – For Antibody
23	Urine Pregnancy Test
24	Example of a Pregnancy Test Urine added to window If patient is not pregnant there is no HCG to bind to vertical line of anti-HCG When labeled anti-HCG is added it has no bound HCG to attach to so when substrate is added no color develops The horizontal line has HCG attached so when labeled anti- HCG is added it is bound to HCG so when substrate is added color develops If patient is pregnant there is HCG to bind to vertical line of anti- HCG When labeled anti-HCG is added it binds to HCG and when substrate is added color develops
25	Sandwich Technique Definition: a same as the non-competitive binding technique for antigen detection. Procedure: 9a commercially prepared antibody is bound to a solid phase 9patient serum containing the unlabeled antigen is then added and will bind with the bound antigen forming Ag-Ab complexes bound to solid phase 9a separation step is necessary to remove the unbound antigen from system 9a labeled antibody is then added in excess which will bind to the multivalent antigen sandwiching the antigen between the two antibodies. 9a separation step removes unbound labeled antibody 9the amount of labeled antibody is then measured 9the amount of labeled antibody is directly proportional to amount of antigen in patient sample
26	One-Step Sandwich Method Advantages: 9extremely sensitive One-step assay: 9a commercially prepared antibody is bound to a solid phase 9patient serum (antigen) and labeled antibody are both added 9an incubation period allows reaction to occur: 9the bound antibody and labeled antibody reacts with different epitopes on the multivalent antibody 9a separation step removes unbound labeled antibody 9the amount of labeled antibody is then measured 9the amount of labeled antibody is directly proportional to amount of antigen in patient sample
27	ASandwich@ Technique – For Antigen
28	Homogeneous vs Heterogeneous Reactions Definition: the fundamental difference is the inclusion of a seperation step in heterogeneous reaction prior to measuring labeled substance whereas the homogeneous reaction does not have a seperation step prior to measuring labeled substance
29	Competitive Radioimmunoassay (RIA) uses the competitive binding technique uses radioisotopes as labels amount of labeled reactant left in system is measured using a radiation detector: ]liquid scintillation counter: ]solid crystal gamma counter: " and $ rays ( rays amount of labeled reactant left in system is inversely proportional to amount of antigen in patient sample
30	Non-Competitive RIA or IRMA also called Immunoradiometric Assay (IRMA) uses the non-competitive binding technique or Asandwich@ technique uses radioisotopes as labels amount of labeled reactant left in system is measured using appropriate radiation detector: amount of labeled reactant left in system is directly proportional to amount of antigen in patient sample
31	RAST Competitive Radioallergosorbent assays: (RAST) also called Aallergen profile@, allergy screen@, or Aallergen- specific IgE quantification@ uses the competitive binding technique where labeled IgE and patient IgE compete for binding sites on a specific allergen bound to a cellulose disc. uses radioisotopes as labels amount of labeled IgE left in system is measured using appropriate radiation detector: amount of labeled IgE left in system is inversely proportional to amount of allergen-specific IgE in patient sample
32	RAST – For IgE
33	RIST Competitive Radioimmunosorbent assays: (RIST) uses the competitive binding technique where labeled IgE and patient IgE compete for binding sites on an anti-IgE antibody bound to a cellulose disc. uses radioisotopes as labels amount of labeled IgE left in system is measured using appropriate radiation detector: amount of labeled IgE left in system is inversely proportional to amount of allergen-specific IgE in patient sample
34	RIST – for IgE
35	Enzyme Labeled Immunoassays uses the competitive binding technique where labeled ligand and patient ligand compete for binding sites on a ligand- specific antibody bound to a solid surface. or uses the non-competitive binding technique where patient ligand binds to sites on a ligand-specific antibody bound to a solid surface. Labeled antibody is then added. Asandwich@ uses enzymes as labels Common enzyme immunoassays include: eEnzyme-linked immunosorbent assays (ELISA) eEnzyme immunoassays (EIA) eEnzyme multiplied immunoassays (EMIT)
36	EIA Enzyme Immunoassays: (EIA) uses the competitive binding technique where labeled ligand and patient ligand compete for binding sites on a ligand- specific antibody bound to a solid surface. may utilize homogeneous EIA E no separation step required or may utilize heterogeneous EIA E a seperation step required to seperated labeled from non-labeled uses enzymes as labels Common applications include: E automation of immunoassays E low levels of drugs detected E low levels of hormones detected
37	ELISA Enzyme-Linked Immunosorbent assays: uses the competitive binding technique where labeled ligand and patient ligand compete for binding sites on a ligand- specific antibody bound to a solid surface. uses enzymes as labels After seperating bound from free labeled and non-labeled ligands an appropriate substrate is added The amount of color complex formed is measured using a spectrophotometer The amount of color developed is inversely proportional to amount of ligand in patient serum
38	ELISA – for antigen
39	EMIT - 1 Enzyme-multiplied Immunoassays: (EMIT) uses the Asandwich@ technique where patient ligand binds with sites on a ligand-specific antibody bound to a solid surface. A labeled ligand-specific antibody is then added which binds with antibody-antigen complex uses enzymes as labels After seperating bound from free labeled antibodies an appropriate substrate is added The amount of color complex formed is measured using a spectrophotometer The amount of color developed is directly proportional to amount of ligand in patient serum
40	EMIT - 2 Enzyme-multiplied Immunoassays: (EMIT) clinical applications include: emeasurement of very small concentrations of an antigen with multiple epitopes emeasurement of immunoglobulins (IgM, IgE etc.)
41	Chemiluminescence Principle: assays that utilize molecules that, upon being excited, emit light through chemical reactions Types of chemiluminescent labels: 9Acridinium esters eupon oxidation they emit a Aflash@ of light 9Luminol eupon oxidation they emit a Aglow@ of light Types of chemiluminescence assays: 9both competitive binding and non-competitive binding reactions 9both homogeneous and heterogeneous protocols may be used
42	Immunofluorescence - 1 Principle: assays that utilize molecules (fluorochromes) that emit light at a longer wavelength than the wavelength of the absorbed light. Types of fluorochromes: 9fluorescein isothiocyanate eabsorbed wavelength = 490 nm eemited wavelength = 517 nm (green) 9rhodamine eabsorbed wavelength = 515 nm eemited wavelength = 546 nm (red) 9flow cytometer
43	Immunofluorescence - 2 Types of immunofluorescence assays: (IFA) 9indirect immunofluorescence assays (IIF) eused to detect circulating autoantibodies in serum 9direct immunofluorescence assays (DIF) eused to detect deposited autoantibodies on tissues Types of immunofluorescence reagents: 9both use commercially prepared fluorescein-labeled mono- specific antibodies
44	DIF Direct immunofluorescence assays: (DIF) 9labeled antibody is added directly to tissue 9incubation period 9washing step to remove any unbound labeled antibody 9viewed using a fluorescence microscope 9positive test shows bright fluorescence at site of ag-ab complexes 9negative test shows no fluorescence 9labeled ag-ab complexes may also be detected by flow cytometry if specimen is fluid
45	IIF - 1 Indirect immunofluorescence assays: (IIF) 9serum samples: enon-competitive binding procedure epatient sample (antibody) added to slide with antigen fixed to it ewashing step removes any unbound patient antibodies elabeled anti-immunoglobulin is added that will bind to ag-ab complex efluorescent ag-ab-labeled ab complex then measured in fluorometer econcentration of antibody in patient serum is directly proportional to amount of fluorescence
46	IIF - 2 Indirect immunofluorescence assays: (IIF) 9tissue samples: epatient tissue sample (antigen + autoantibody complexes) fixed to slide elabeled anti-immunoglobulin is added that will bind to ag-ab complex efluorescent ag-ab-labeled ab complex then analyzed using a fluorescence microscope ethe following observations are made: jpattern of deposits (granular or linear) jextent of staining (focal or diffuse) jlocation of deposits (nuclear, basement membrane, mitochondria, etc.)
47	OSF - SLE On page 284AOne Step Further@ presents a more in-depth discussion of Systemic Lupus Erythematosus and it=s detection. This presentation is contained on a separate slide presentation called A One Step Further #15" The student may call up the slide program OSF-15 later or click on the arrow below to view slides now.
48	End of Labeled Immunoassays Press the ESC key to end program
49	One Step Further Hasimoto=s Thyroiditis OSF - 14 Page 276 7click arrow to return to main program
50	Hashimoto=s Thyroiditis - 2 Mechanism of disease: eautoimmune disorder seen most often in women and is leading cause of thyroiditis (inflammation of thyroid gland). ethese autoantibodies are directed against several thyroid- specific antigens ewhen antibodies attach to antigen an inflammatory response is initiated and the disease progresses to hypothyroidism Autoantibodies of disease: eanti-thyroglobulin (antiTg) Antibodies against precursors of the thyroid hormones (thyroglobulins) eanti-thyroid peroxidase (anti-TPO) also called Aanti-microsomal antibodies@ directed against thyroid peroxidase antigen
51	Hashimoto=s Thyroiditis - 3 Other diseases with anti-Tg and anti-TPO present: ehyperthyroidism ethyroid tumors epernicious anemia - lack of intrinsic factor leads to vitamin B₁₂ deficiency elupus erythematosus esome healthy individuals may test positive
52	Hashimoto=s Thyroiditis - 4 7click arrow to return to main program
53	One Step Further Systemic Lupus Erythematosus OSF - 15 Page 284 7click arrow to return to main program
54	SLE - 2 Mechanism of disease: eautoimmune disorder in which there is a non-organ specific hyperactivity of the immune system ethese autoantibodies react with a variety of tissue antigens causing ag-ab complexes to be formed ewhen ag-ab complexes are deposited on tissues it results in destruction of that tissue Autoantibodies of disease: eantibodies are polyclonal Antibodies to native DNA (anti-DNA) or also called double-stranded DNA (dsDNA) Antibodies to Smith antigen (anti-Sm Antibodies are of the IgG and IgM class
55	SLE - 3 Causes of disease: emay have viral origin when viral stimulation of host DNA leads to production of anti-DNA antibodies emay have genetic link: HLA-DR2 - anti-dsDNA formed HLA-DR3 - anti-SS-A and anti-SS-B formed HLA-DR4 & HLA-DR5 - anti-Sm & anti-RNP formed Clinical findings: earthritis eskin rashes enephritis epulmonary disorders ecardiac disorders eoptical disorders eG.I. disorders eCNS disorders
56	SLE - 4 Immunologic testing: epatients have elevated " and ( globulins epatients have decreased levels of albumins and complement proteins C3 and C4 epatients have high titers of anti-Sm and anti-dsDNA epatients display nephrotic disorders due to ag-ab complexes being deposited on basement membranes of glomerulus epatients display skin rashes due to ag-ab complexes being deposited on dermal-epithelial junctions
57	SLE - 5 7click arrow to return to main program