1	CLSC320 Principles of Immunology Diagnostic Laboratory Immunology Program for Clinical Laboratory Science Unit - 11 Precipitation Techniques
2	Unit 11 Guidelines Reading assignment: Pages 228 - 252 of textbook Learning objectives: Those listed on page 229 of textbook Key terms: Those listed on pages 229 - 230 of textbook
3	Introduction to Precipitation Techniques Definition: Immunoprecipitation is the formation of insoluble Ag-Ab complexes called Aprecipitins@, when soluble antigen is mixed with it=s specific antibody. Factors that must be considered when performing immunoprecipitation techniques: Urelative concentrations of antigen and antibodies Uhydrogen ion concentration (pH) Uionic strength of solution or reaction media Uantibody affinity and avidity
4	Precipitation Procedures Immunodiffusion techniques: Immunoelectrophoresis techniques: Light Scattering Immunoassay techniques: Usingle immunodiffusion (Oudin) Udouble immunodiffusion (Ouchterlony) Uradial immunodiffusion Uimmunoelectrophoresis (IEP) Uimmunofixation electrophoresis (IFE) Uelectroimmunodiffusion \countercurrent electrophoresis \rocket electrophoresis Uisoelectric focusing Uimmunoturbidimetric methods Uimmunonephelometric methods
5	Definition of Immunodiffusion Definition: Immunodiffusion is the movement of antibody molecules and antigen molecules within a support medium When the two reactants meet insoluble Ag-Ab complexes are formed (precipitins) which are visible and fixed at site of precipitation Antigen Antibody precipitin
6	Double Immunodiffusion - 1 Also called Ouchterlony procedure UPattern of Identity Continuous precipitin lines that merge and form an arc indicate antibodies are precipitating identical epitopes antibody antigen 1 antigen 2 precipitin line
7	Double Immunodiffusion - 2 UPattern of Partial Identity fusion of precipitin lines indicate antibodies are precipitating identical epitopes common to both antigens while the spur indicates an additional epitope not common to both antibody antigen 1 antigen 2 precipitin line precipitin spur
8	Double Immunodiffusion - 3 UPattern of Non-Identity fusion of precipitin lines indicate antibodies are precipitating non-identical epitopes of antigens antibody antigen 1 antigen 2 precipitin line precipitin line
9	Precipitation Techniques - 5 Immunodiffusion Techniques Double Immunodiffusion Technique (Ouchterlony): UPractical application: The Alberta Health Dept. would like to confirm that the chicken chop suey from Wings cafJ is really chicken. chicken chop suey anti-rabbit anti-cat anti-dog anti-chicken precipitin line precipitin line
10	Double Immunodiffusion – Clinical Applications eMixed Connective Tissue Disease an autoimmune disorder in which antibodies react with a variety of tissues and looks like: SLE - nuclear proteins scleroderma - blood vessels & connective polymyositis - muscle eSystemic Lupus Erythematosus an autoimmune disorder in which antibodies react with a variety of tissues
11	Double Immunodiffusion – Technical Errors eAlteration of precipitin lines by: 1.excessive condensation in wells 2.improper filling of wells 3.incubation in tilted position efalse results due to misidentification of wells efalse negative results due to under incubation efalse results due to incorrect interpretation of pattern einvalid controls or standards used
12	Single Radial Immunodiffusion Also called Oudin procedure: One of the reactants is incorporated into the medium and is in fixed position and concentration while the other reactant diffuses through the medium from wells cut into medium. agar with Ab=s to Ag being tested Standards of differing concentrations added to wells a - d a b c d precipitin rings the larger the ring the greater the conc. of Std.
13	Standard Curve for Oudin Test A standard curve can then be developed based on concentration vs ring diameter. 1 2 3 4 0 b c d a Diameter (mm) Concentration (mg/dL) X X X plot ring size for each concentration join dots to form std curve
14	Calculation of Conc. – Oudin Test - 1 Patient samples now can be run using same agar media Patients a through d added to individual wells a b c d The size of each ring is then plotted on std curve to determine concentration of each patient=s antigen
15	Calculation of Conc. – Oudin Test - 2 A standard curve can then be used to convert ring size to concentration 1 2 3 4 0 1 2 3 0 X X X Diameter (mm) Concentration (mg/dL) let=s say patient c has a ring size of 3 mm patient c has a conc. of 3 mg/dL let=s say patient a=s ring size = 2.5 mm = 2.3 mg/dL let=s say patient b=s ring size = 2 mm = 2.0 mg/dL let=s say patient d=s ring size = .5 mm = 0.5 mg/dL
16	Two Methods of Determining Conc. eEnd-Point Method (Mancini): eKinetic Method (Fahey & McKelvey) 1.antigen allowed to diffuse until completion - about 24 hours 2.the ring diameter is directly proportional to concentration of antigen 3.standard curve used to convert ring diameter to concentration 1.antigen allowed to diffuse for limited time - about 18 hours 2.the ring diameter is proportional to the log of antigen concentration equivalence point is reached equivalence point is not reached
17	Clinical Applications & Errors Clinical Applications: Identification and quantification of serum proteins: -immunoglobulins (IgG, IgM, IgE, IgD, IgA) -"₁-antitrypsin -transferrin -complement components (C3, C5, etc.) Technical errors: incorrect filling of wells specimen contamination wrong media or outdated media incorrect construction of standard curve incorrect incubation time or plate position incorrect measuring of ring size or calculations
18	Electrophoretic Techniques - 1 General concept: By applying voltage through a support medium it is possible to separate complex protein mixtures Types of support media used in clinical applications: Upaper Uagarose gel Ucellulose acetate Factors influencing protein molecule migration: Usurface charge on molecule Umolecular weight of molecule Usize of molecule Umedium characteristics Ubuffer characteristics (pH) Utemperature
19	Electrophoretic Procedure Serum, urine, or CSF is applied to support medium and voltage is applied point of application anode (+) cathode (-) a stain is added that reacts with separated proteins to form a visible band of color the strip is then placed in a densitometer which converts each band into a peak based on intensity of color and width of band
20	Densitometer Tracing – Normal Serum anode (+) cathode (-) ( $ "₂ "₁ albumin
21	Densitometer Tracing – Monoclonal Gammopathy anode (+) cathode (-) ( $ "₂ "₁ albumin Waldenstrom=s macroglobulinemia Multiple myeloma
22	Densitometer Tracing – Polyclonal Gammopathy anode (+) cathode (-) ( $ "₂ "₁ albumin chronic infection chronic inflammation chronic liver disease if on CSF it may indicate oligoclonal bands as seen in multiple sclerosis
23	Electrophoretic – Technical Errors UArtifacts from hemolyzed specimens causes increases in: -"₂-globulin peak -$-globulin peak Upoor or excessive migration due to: -wrong amount of time allowed for test to run -wrong amount of voltage applied -wrong pH due to bad buffer Uexcessive sample applied will cause: -increased peaks -cross contamination of adjacent peaks Uincreased peak in $-globin area may be due to: -presence of fibrinogen may be mistaken for increased IgA levels
24	OSF – Waldenstrom’s Waldenstrom=s Macroglobulinemia On pages 239 - 241AOne Step Further@ presents a more in- depth discussion of the Waldenstrom=s Macroglobulinemia. This presentation is contained on a separate slide presentation called A One Step Further #10" Cytokines The student may call up the slide program OSF-10 later or click on the arrow below to view slides now.
25	OSF – Multiple Sclerosis Multiple Sclerosis On pages 243 - 244AOne Step Further@ presents a more in- depth discussion of Multiple Sclerosis. This presentation is contained on a separate slide presentation called A One Step Further #11" The student may call up the slide program OSF-11 later or click on the arrow below to view slides now. Cytokines
26	Immunoelectrophoresis - Concepts Utypically performed as confirmation when a monoclonal component is detected on protein electrophoresis. Uallows for characterization of monoclonal proteins by identifying: -specific heavy-chain classes -specific light-chain components Ua two-step process that involves: -electrophoretic separation of proteins in serum or urine -immunodiffusion/precipitation of separated proteins with antiserum
27	Immunoelectrophoresis - Procedure Userum or urine placed in antigen wells in a test gel. Utheir proteins are then seperated using protein electrophoretic technique. Uantisera (monospecific reagent antibodies) then placed in trough in gel. Uantisera and protein fraction diffuse towards each other and form precipitin lines when equivalence point is reached.
28	Figure - Immunoelectrophoresis gel plate Ag well antisera trough serum added anode (+) cathode (-) separated proteins separated proteins
29	Immunoelectrophoresis - Precipitin monospecific antisera added -incubation in humid chamber for specific time precipitin lines form -position of precipitin lines compared to control for identification -increased in size of precipitin line indicates abnormal production of antibodies -decreased in size of precipitin line indicates immunodeficiency
30	IFE – General Concepts IFE = Immunofixation Electrophoresis UUsed as an alternative to immunoelectrophoresis UA two step procedure as is IEP. UDiffers from IEP in: \the antisera is directly overlayed onto gel instead of being put in a trough \more sensitive \shorter incubation time (2 hours compared to overnight) \more expensive and labor-intensive
31	EID & EIA - Concepts EID = Electroimmunodiffusion UCountercurrent electrophoresis \similar to that of double immunodiffusion with the addition of an electric current to aid in seperation of proteins EIA = Electroimmunoassay (Rocket electrophoresis) \depends on the creation of different charges of antibody and antigen at selected pH \as antigen moves through gel several precipitin bands appear forming Arockets@.
32	Isoelectric Focusing General concept: UBy creating a pH gradient within the medium immunoglobulin molecules (amphoteric) will move in an electrophoretic system to a point where their isoelectric point is equal to the pH of medium amphoteric molecule = molecules that can act as either a base or an acid depending on pH of medium in which they are suspended. Clinical application: U identification of oligoclonal bands as in: \multiple sclerosis \viral encephalitis \cerebral infarction \AIDS
33	Light Scattering Immunoassays General concept: Uwhen ag-ab complexes are formed in a solution they form immunoprecipitates which have the ability to scatter a light beam that passes through the solution. Turbidimetric application: U detectors set at 180^o angle measures reduction in light Nephelometric application: U detectors set at 90^o angle measures increases in light in both techniques standard curves are constructed to convert absorbance to concentration in turbidimetry the absorbance is directly proportionate to concentration so std curve is linear and fewer standards needed in nephelimetry the standard curve is non-linear and requires calibrators
34	End of Precipitation Techniques Press the ESC key to end program
35	One Step Further Waldenstrom=s Macroglobulinemia OSF - 10 Pages 239 & 241 7click arrow to return to main program
36	Waldenstrom=s - 2 Classification: ea monoclonal gammopathy with malignant lymphoproliferation Monoclonal gammopathies: euncontrolled proliferation of a single clone or line of plasma cells ealso called Aplasma cell dyscrasia@ eplasma cell clone produces a homogenous monoclonal protein (M-protein) ein the case of Waldenstrom=s the clone produces a homogenous monoclonal protein of IgM class immunoglobulins
37	Waldenstrom=s - 3 Mechanism of Disease: ecause unknown egenetic predisposition may play a role emainly seen in older individuals epresents with the following features: Žmalignant proliferation of B cell line Žincreased plasma cell population Žhigh levels of IgM produced Žweakness, fatigue, nose & gum bleeds, and GI bleeds leads to anemia Žincreased infections Žweight loss Žincreased blood viscosity Žmay develop neurological and cardiac problems
38	Waldenstrom=s - 4 Laboratory Findings: epresence of M-protein on serum electrophoresis eserum & urine characterization of M-protein using: Žimmunofixation test (IFE) - one of choice Žimmunoelectrophoresis test (IEP) equantification of serum IgM levels using: Žnephelometry procedures eM-protein of Waldenstrom=s may be: Žin serum: wIgM heavy chains weither kappa or lambda light chains Žin urine: weither kappa or lambda light chains wfree light chains are called ABence-Jones proteins@
39	Waldenstrom=s - 5 Testing Protocol: ŽScreening tests Serum Protein Electrophoresis (SPEP) monoclonal bands present? NO no monoclonal gammopathy present YES Perform confirmatory testing to next slide
40	Waldenstrom=s - 6 Testing Protocol: ŽConfirmatory tests Urine and serum specimens CHARACTERIZATION OF MONOCLONAL BAND QUANTIFICATION OF IMMUNOGLOBULINS Serum IFE ID type of heavy chains and light chains Urine IFE Confirm Bence-Jones protein Serum Neph determine conc. of Ig=s Urine Neph determine conc. of light chains
41	Waldenstrom=s - End 7click arrow to return to main program
42	One Step Further Multiple Sclerosis OSF - 11 Pages 243 & 244 7click arrow to return to main program
43	Multiple Sclerosis - 2 Mechanism of Disease: ecause is unknown ebelief is that genetic and environmental factors play a role ŽGenetic factors: these two HLA antigens are present in patient=s with MS ~DRw15 ~DQw6 ŽEnvironmental factors: ~inflammatory immune response to bacterial and viral infections may trigger autoimmune response seen in MS patients e80% of MS patients have IgG produced in CSF that is directed against basic protein of myelin sheath. This results in demyelination and formation of lesions called Aplaques@ in white matter of brain and spinal cord edemyelination may cause: visual distrubances lack of locomotor coordination
44	Multiple Sclerosis - 3 Laboratory findings: eit is important to determine source of IgG in CSF Žis it produced in the spinal cord Žis it produced elsewhere and has crossed the blood- brain barrier etwo assays used to determine origin of IgG in CSF: Žoligoclonal banding technique: ~protein electrophoresis of CSF shows multiple bands in the ( region ~protein electrophoresis of serum shows multiple bands in the ( region ~if isoelectric focusing used: qCSF shows band pattern in ( region qserum shows no band pattern in ( region
45	Multiple Sclerosis - 4 Laboratory Findings: continued etwo assays used to determine origin of IgG in CSF: cont=d ŽCSF IgG index technique: ~this is a calculation that uses the ratio of IgG to albumin in both serum and CSF CSF IgG index = IgG_CSF ) albumin_CSF IgG_serum ) albumin_serum ~interpretation of results: q0.0 - 0.77 q> 0.77 serum IgG in CSF IgG produced in CSF
46	End of Multiple Sclerosis 7click arrow to return to main program