lab techniques Flashcards
(24 cards)
what are immune mediators
- antigens
- antibodies
- cytokines
- cells
what does the concentration of immune mediators tell us
information of the disease state - mechanism, diagnosis, response to treatment
what occurs with a blood group assay
- dilute blood sample
- add antibodies against erythrocyte antigens (A, B ,Rh)
- agglutination occurs to determine blood group
what does a radioimmunoassay do
- competitive antibody binding assay
- cold insulin competes with hot insulin
- higher 125 I insulin means less normal insulin and type 1 diabetes
what is the disadvantage of radioimmunoassay
radiolabel is hazardous
what is the advantage of radioimmunoassay
faster than chromatography based methods
what does a low sample of insulin present as in an radioimmunoassay
- low conc of 125I labelled insulin
- high radiolabelled signal
what does a high sample of insulin present like in an radioimmunoassay
- high conc of 125 I labelled insulin
- low radiolabelled signal
what does ELISA stand for
enzyme linked immunosorbent assay
what are the types of ELISA
direct, indirect, competitive and non-competitive
what does an indirect, non competitive ELISA do
- measures specific antibody concentration
- antigen adsorbed to well surface
- blocking solution blocks non-specific binding
- add primary antibody in excess and wash off unbound
- add conjugated antibody in excess
what is a conjugate in ELISA
- an enzyme - catalyses further reaction
- florescent molecule - spectrophotometry
how is an indirect, non competitive ELISA used in clinical practice
- detection of antibodies in response to infection
- diagnostic for HIV
- detection of antibodies for immunisation eg. Hep B
why use a indirect ELISA over direct
- every antibody labelled individually
- saves time, labour costs and increases safety
- increases sensitivity
how do you use ELISA to measure antigen concentration
- antigen filling between two antibodies (sandwich)
- first captures with a high affinity and next recognises epitope
what does western blotting do
- proteins have a + and - charge
- separates protein sample on SDS-polyacrylamide gel by electrophoresis
what does western blotting tell us
- distance travelled - estimation of molecular weight
- amount of chemiluminescent signal allows estimation of abundance
how is western blotting used in clinical practice
- conformation test for anti-HIV antibodies
what are rapid/ point of care tests used for
- detection of antibodies for HIV, hep C, covid-19
- detection of antigen for malaria, ebola, influenza, strep, HCG
what does immunofluorescence show
visualisation of tissue, structures, cells and sub- cellular components
how does immunofluorescence work
- need to be able to detect if antibodies react with cellular antigens
what does flow cytometry do
Counts cells based on size,
contents, and cell surface
expression
what are the steps in flow cytomerty
1- prepare single cell suspension
2- make fluorescently labelled antibodies
3- stain patients leukocytes
4- count on Flow cytometer