antibodies as diagnostic tools Flashcards
describe antibody structure
Fc constant region
Fab variable regions
what’s are the 2 types of reporter?
enzyme: bound to antibody, normally substrate is colourless but when antibody binds to antigen, substrate product changes to coloured product
magnetic beads: useful for pulling out particular cell type from a sample of cells
when are antibodies produced in patients?
autoimmune disease
defence against infection
what are the 3 types of manufactured antibodies?
anti sera: polyclonal, collecting antibodies from an animal which had antigens inserted into
monoclonal antibody: B lymphocytes and myeloma cell line are fused, theses hybridoma are used to harvest antibodies
genetically engineered antibody: V segments are inserted into bacteriophage and library of bacteriophage are used to screen plates with antigen mobilised in it
what is the benefit of using antibody in diagnostic?
specificity
what are the uses of manufactured antibodies?
therapeutic - prophylactic protection against microbial infection
- anti-cancer
- removing T cell from bone marrow grafts
- block cytokines activity
diagnostic - blood group serology
- immunoassay
- immunodiagnosis: infectious disease, autoimmunity, allergy, malignancy
describe therapeutic monoclonal antibody nomenclature
- omab: mouse
- imab: partly human
- umab: human
how does ELISA work?
enzyme linked immunosorbent assay
wells covered with particular antigen you are trying to measure
reporter = enzyme
colourless substrate added
if antibodies bind to antigen, substrate broken down to coloured product
unbound antibody washed away
measure absorbance of light
what are the immunological concerns over immune complexes?
= antibody bound to antigen
size indicates ration of antigen to antibody
large -> easily recognised by immune system -> activate platelets and neutrophils -> mediators affecting endothelial layers released
small -> only activate complement when bound to surface
-> pass through basement membrane -> gets trapped in subendothelial layer -> activate complement --> kidney function damage
inflammation, serum sickness
glomerulonephritis, deposition at other sites (skin, joint, lungs)
how is serum immunoglobulin level tested for immunodeficiency?
serum electrophoresis
healthy serum have diffuse smear against gamma globulin region
active immune response serum have more gamma globulin so darker - polyclonal expansion
sharp single band of serum indicates monoclonal expansion of B cells
how is lymphocyte subsets tested for immunodeficiency?
flow cytometry
specific marker of each lymphocyte subsets is targeted by antibodies
how do you measure cell population?
add different monoclonal antibodies labelled with different fluorescent dye into cell mixture
cells pass through laser beam where fluorescent are detected
each cells are categorised on the basis of its fluorescence
describe HIV infection timeline
CD4 T cell count initially goes down - increased viral load
after a few weeks CD4 T cell count goes up - sustained viral load -> antiretroviral therapy
eventually CD4 T cell count goes very low, quick increase of viral load -> opportunistic infection -> death
how is HIV diagnosed and monitored?
HIV antibody test positive
CD4 T cell count and viral load
how is CD4 T cell count and opportunistic infection linked?
different infections as CD4 count goes down
when very low, MAC infections (mycobacterium avium complex - environmental bacterium that is everywhere and well dealt with by healthy people)
