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18 Cards in this Set
- Front
- Back
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clinical lab tests involving Ab & Ag rxns for identification, detection or quantitation of proteins, polysaccharides & other analytes can be placed into 1/4 categories:
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precipitation
agglutination labeled Ab |
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precipitation is analysis of
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soluble proteins/analytes
C reactive protein, immunoglobulin levels, complement components, hormones & many other serum urine proteins |
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agglutination is analysis offf
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particulate or cellular rxns
RBC typing, rheumatoid factor, RhD Abs (hemolyytic disease newborn), many latex immobilized Ags |
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labeled Ab
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isualization, localization of Ag in tissues or on cells (fluorochromes)
- kidney pathology & flow cytometry signal amplification of primary rxn (enzymes & radioactive labels) - Enzyme linked immunosorbant assay (ELISA), immunoblotting& radio-immunoassays |
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preciptation rxns
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Ab crosslinks epitopes on soluble proteins
requires Ag is multivalent : many epitopes forAb binding crosslinking decreases solubility of protein as it forms Ag-Ab complexes; solution increases from clear through various stages of turbidity as rxn takes place Ag-Ab at equivalence forms macro-molecular complexes that preciptate out of soln rxn detected at various stages by various means for quantifying Ag |
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preciptation assays for clinical lab have been adapted for liquid & gel matrixes
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immunodiffusion
immunoelectrophoresis immunofixation nephelometry |
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Equivalence curve Ag-Ab complexes
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quantitative equivalence curve: titrating/diluting Ab
concept, Ag-Ab binding not covalent & reversible Ab-serum or commercial Ab prep is diluted Ag (containing multiple epitops) added in equal concentrations to Ab dilutions resulting in differing degress of Ag-Ab complex formation Ag-Ab complexes are formed into 3 zones can generate same type of curve by titrating/diluting Ag & adding Ab to each tube in constant amount |
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Ag-Ab complexes formed in 3 zones
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zone of Ab excess: Ab molecules out# available epitopes no preciptating complexes form
- prozone effect equivalence zone: both Ag & Ab are at concentrations that result in maximal lattice formation - macro-molecular complexes zone of Ag excess: available epitopes out# Ab-binding sites - no precipitating complexes form pt at which cross linking of particulate or soluble Ag is no longer observed is called precipitin titer |
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radial immunodiffusion
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Ab (commercial) added to melted solid matrix (agar), poured & solidified
- determines specificity of test solution containing suspected Ag (serum,spinal fluid, urine) added to wells punched in agar Ag diffuses into agar equally in all directions (radial) & forms precipitate when equivalence occurs some distance from well area within precipitin ring closest to well is a zone of Ag excess area outside the precipitin ring is in Ab excess diameter of precipitin ring is directly proportional to initial Ag concentration & quantified from standard curve |
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immunofixation electrophoresis
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supportive diagnosis of monoclonal gammopathies from multiple myeloma of plasma cells
-- produce lots of 1 specific Ab |
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nephelometry
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Ag-Ab complexes form turbidiy in solution and scatter light
rate of light scatter when Ag-Ab complexes form (turbidity) used to rapidly measure concentration of analyte in pts sample using a nephelometer standard commercial preps of appropraite Ab & analyte of known concentrations are used to form a standard curve & calibrate the nephelometer light scattered is proportional to concentration of analyte Ag in mix, usually serum, urine or other body fluid |
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agglutination
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particulate Ags such as erythrocytes, bacteria or Ag-coated latex beads are cross linked by Ab
positive test for particular Ag-Ab rxn is visualized as clumping 2 types : direct & indirect |
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direct agglutination
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usually detects IgM Abs
1 step directly causes agglutination blood group Ags Rheumatoid factor |
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indirect agglutination
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need 2nd Ab (commercial) to indirectly cause agglutination
- RhD Ab in RhD negative mom usually detects other IgG Ab isotypes |
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Coombs test
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cellular Ags: RBC with ABO, RhD Ags on cell surface
= Ab in pts serum to cellular Ags (RBC) - requires commercial anti-IgG to agglutinate cells after Ab binding - RhD (indirect coomb's: 2 step rxn) = agglutination (clumping) positive for Ab to test Ag Ab: cells coated with bound Ab or Ab against Cells = Ab already bound to cells (RBC) - requires commercial Ab to agglutinate - RhD (direct coomb's 1 step rxn) |
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Rheumatoid factor
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occurs in serum of pts with RA and some other autoimmune diseases
an Ab against Fc region ofpts own IgG forms IC's in sera, synovial fluid & synovial membranes is most often IgM latex beads coated with denatured IgG; RF will crosslink IgG & directly agglutinate the beads |
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direct coombs
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testing for Abs from Rh-neg sensitized mother bound to fetal RBC
get fetal RBC & add anti-human Ig agglutination is positive direct coombs for HDN Anti-RhD Ab (moms) bound to fetal RBC positive for HDN |
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indirect coombs
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testing whether Abs to RhD are present in serum of a mother potentially previously sensitized from first birth
incubate serum with RhD (Ag) positive and allow Abs to bind to RBC: step 1 add commercial anti-human Ig: step 2 agglutination is positive for anti-RhD in moms serum Anti-RhD Ab in serum. 2nd RhD positive fetus high risk of HDN |
