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32 terms

Principles of Seralogical Testing

Principles of Aggulation
-antibodies/antigens absorbed onto surface of carriers
ex bacterial cells
Latex Agglutination Test /(AGGULATION)
-antibody/antigen bound to latex bead
Clinical Sig- C-reactive protein(C4)
Anti-Streptolysin O Antibody Test/(AGGULATION)
-tests against Streptolysin O, this causes impetigo and cellulitus
-skin infection can cause low ASO titer
-Low ASO can be caused due to frequency of strep throat infection
Flocculation Test /(AGGULATION)
-results in formation of precipitate of fine particles/ AGGULATION TEST
-causes Syphilis VDRL/RPR
Hemaggultination Methods (AGGULATION)
-antigens can be natural or bound
-gently shake test tube and view with LOWER portion of magnifying glass
-tests for HIV I II
Grading Agglutination
Mixed field- free floating cells red supernatant
Weak-tiny aggregates
+1- Few aggregates
+2- Medium aggregates, clear supernatant
+3 Several Large Aggregates
+4 One solid aggregate
Precipitation Method
-soluble antigen/antibody produce insoluble complex
-not widely used in general lab setting
Double Immunodiffusion Test/Ouchterlony DDT (PRECIPTIATION)
-ab/ag form immune complex that precipitate
Identity- bands form smooth arc
Non-Identity: lines cross
Partial Identity: lines merge in spur formation
-combines diffusion with electrophoresis
-agarose gel
Countercurrent Immuno-electrophoresis(CIE)
-ph chosen so AB are + charge AG -
used for Radioimmunoprecipitation and Antinuclear precipitation
Rocket Electrophoresis
-pH added to inhibit AB migration
-rocket height proportional to antigen concentration
Immunofixation Electrophoresis(IFE)
-two stage procedure
-specimen applied to six different positions
-incubate plate for 10 mins
-use protein sensitive stain for identification of Immunoglobins
Labeled Immunoassay
-used for ab/ag that are small in size or low in concentration
-presence determined indirectly to detect whether binding takes place
Radioimmunoassay RIA
-relies on the principle of competitive binding
-radioactive analyte is in excess
HEP A IgM/ Hep A Ag
Immunoradiometric Assay IRMA
-same method as RIA, but uses 2 monoclonal antibodies
-has faster reaction rate and increased sensitivity
Defined as *light emission caused by product of reaction
-*supplanted most RIA
-excellent dynamic range
-no radiation
-nonselective excitation and instability removed
Chemiluminesence Formats
Competitve Immunoassay-fixed amount of antigen
Sandwich Immunoassay- uses multiwalled plate
Enzyme Immunoassays Types
-Alkaline Phosphatase
-Horseraddish Peroxidase
Enzyme Immunoassay(EIA) Principle
-uses plastic beat/plate
-measured spectrophotometrically
Immunofluorescent Techniques
-uses fluorescent molecules as labels
Direct Fluorescent Assay: Best suited for Antigen
Indirect Fluorescent: used for ab and ag
techniques are extremely specific and sensitive
Direct Immunofluorescent
-AB coated with fluorescent tag*
-Antigens typically visualized as bright apple green/orange yellow
Indirect Immunofluorescent Assay
-involves reaction of patient serum w/ known antigen
-ANA's frequently assayed using this method
Antinuclear Antibody Screening Test
-Antigen substrate from tissue containing nuclei is fixed to slide
Complement Fixation
Test Has two components
-Indicator system(sheep rbc)
-Known antigen and patient serum
-binds complement to ag/ab complex
Complement Fixation Clinical Sig
-hemolysis of sheep cell indicate lack of antibody
tests for herpes simplex lol
PCR Test
-amplifies low levels of DNA to higher quantities
-relies on DNA-copying enzymes under a hot temp
PCR Steps
Step 1. Denature DNA(165deg F)/ 75-90 C
Step 2. Primer Annealing cool vial to 55 so primer can anneal
Step 3. Extension :Raise temp to 75C
PCR Significance
-30 cycles create 1 billion copies of a single DNA
-can be reversed to create RNA(RT-PCR)/Reverse Transcriptase
-detects gene mutations for cancer
Automated Procedures
-Flow Cytometer
-Fluorescent Polarization Immunoassay
-Immunoradiometric Assay(IRMA)
Flow Cytometer
Combination of
-fluid dynamics
-laser science
-fluid flurochrome conjugated monoclonial antibodies for real
Flow Cytometer major applications
-Identification of Cells
-Cell sorting before further analysis
Fluorescent Polarization Immunoassay(FPIA)
-perform microparticle EIA and ion capture (enzyme immuno assay)
-allows for high and low molecular weight analyte measurement
-Clinically applied to endocrine function and fertility