Immunodiagnostics 2 Flashcards
Simple tests
full blood count and protein electrophoresis
Specialized tests
immunoassays, immunofluorescent and immunophenotyping. Tests for type of antigen and specific antibody presence
ELISA Outline
Enzyme Linked Immunosorbent assays. 2 Types: antigen and antibody detection
Antibody Detection method
Can test for 96 different types of antigen. Known antigen is bound to well, add patient serum to same well and add antibody containing horse-radish peroxidase and then add TMB substrate. If patient sample is present solution should turn blue
Why does sample turn blue in presence of antibody
Secondary antigen containing horse-radish peroxidase binds to Fc gamma 3 binding site on
primary antigen. The binding of the secondar antigen horse-radish that oxidises TMB from colourless to blue colour
Antibody Detection Applications
Allergy identification, autoimmunity (eg coeliac disease), cancer and chronic infection
Antigen Detection Method
Add patient serum to well, add commercial primary antibody to well, add commercial secondary antigen (containing horse raddish peroxidase) to well and then add TMB substrate. If antigen is present in patient sample well will turn blue
Immunochromatographic Strip Outline
Antigen test method. Patient’s sample is taken by swab, diluted and then added to paper. at indicated lines ther are antibodies. As liquid medium carries sample up paper antigens attach to antibodies forming coloured lones. Control line = microbes part of noses normal flora
Antigen Detection Application
Rapid testing (eg SARS), drug and pregnancy tests (human chronic ghondoprotein detection)
How autoimmunity occurs
Some host cell receptors are similar in shape to antigens. Autoimmunity occurs when antibodies act where they shouldn’t. Can be specialised or systemically
Autoantibodies Outline
Antibodies that attack self antigen. 2 types: anti-nuclear antibodies (ANA) and anti-neutrophil cytoplasmic antibodies (ANCAs). 1st line tests of autoantibody presence (general)
ANA Immunofluorescence Method
Smear human epithelial cells on slide, patient sample is added at different dilutions. If ANA is present in binds to epithelial nucleus. Add fluorescein labeled antibody to slides. Distinct patterns are observed by microscopy
Homogenous ANA Pattern Outline
Rheumatoid arthiritis, systemic lupus erythematosus
Speckled ANA Pattern Outline
systemic lupus erythematosus and Sjorgens syndrome
Centromere ANA Pattern Outline
progressive systemic sclerosis
Nucleolar ANA Pattern Outline
systemic lupus erythematosus and progressive syndrome sclerosis
Tests that follow ANA
Anti-ENA panel, anti- dsDNA, anti-centromere or antihistone
ANCA Immunofluorescence method
Commercial neutrophils mixed with commercial serum is coated on slides, Patient sample added to slide and commercial antibodies with fluorescent tag added
What does ANCA bind to
myeloperoxide (MPO, perinuclear (passes membrane)) and proteinase 3 (P3, cytoplasmic, near membrane)
Immunophenotyping Outline
Diagnose/classify leukemia or lymphoma and immunodeficency. Detects presence/absence of specific white blood cells. Done by fluorescence activated cell sorting
Immunophenotyping Method Outline
White blood cells suspended in physiological solution. Commercial antibodies tagged with fluroescent markers are added. Antibodies bind to corresponding white blood cells. Cells forced through multiple laser beams. Cell size and granuality are measured. Type and quantity of fluroescent tag is measured. Resulta are quantified cells/sec
Immunophenotyping Example
CD4 levels are low in HIV patients
