Principles of Seralogical Testing

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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

Immuno-Electrophoresis(IEP)

-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
-PRIMARLY USED FOR MONOCLONAL ANTIBODIES

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

Chemiluminescense

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
-Beta-Galactosidase

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
-optic
-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

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