Review of laboratory assignments (Heeringa)
14 important questions on Review of laboratory assignments (Heeringa)
What is immunohistochemistry a tool for?
Anti-Neutrophil Cytoplasmic Autoantibodies (ANCA).
What is the result of ANCA-associated small vessel vasculitidis?
What are the two primary antigenic targets for ANCA?
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In what kind of patients are ANCA frequent?
Why is ANCA testing important?
Explain indirect immunohistochemistry for ANCA.
Primary antibody = Sample
Secondary antibody is Labeled with horseraddish peroxidase (detection)
Colour development HRP+H2O2+ AEC= brown precipitate
(Q: What is the purpose of this incubation step?) 15 min incubation with H2O2
What is the titer in immunohistochemistry?
What is the advantage of immunofluorescence over immune peroxidase for diagnosis?
With anti-MPO autoantibodies, a (peri)nuclear staining pattern is seen on ethanol-fixed neutrophils. On paraformaldehyde-fixed neutrophils, however, this changes into a cytoplasmic staining pattern. Explain this difference (hint: MPO has an isoelectric point of >11)
At pH below the IP proteins have a net positive charge
At pH above the IP proteins have a net negative charge
MPO: IP of >11 à very positively charged
• Ethanol fixation allows proteins to ‘move’à strongly cationic MPO translocates to the nucleus (net negative charge)
• Formalin fixation cross-links lysin residues in proteins which prohibits protein movement.
What is indirect ELISA?
• detection of immunoglobulins (IgG, IgM, IgA, IgE, or subclasses) specific for the antigen.
2: The ELISA performed here is an indirect ELISA. How could you make the ELISA test more sensitive and specific?
When is an antibody no longer effective for viruses?
What is the difference between pleiotropy and redundancy of cytokines?
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