Immuno: Techniques

methods to determine affinity of ag ab interaction

• equilibrium dialysis can be used to measure ab affinity for ag

  • older, easier, cheap

• separate the membrane eventually reach equilibrium

• if ab is on one side = ag will stick to ab too big to go back not at equilibrium

• how much ag will ab bind

Mice

• ingest mouse with ag

  • different epitopes

  • want one clone

• isolate spleen cells from mice seperate out

  • plasma cells

  • myeloma cells

• hybridize

  • hybridomas: tumor cells from mouse immortal put into media make the membrane fluid and fuse together

  • select for ones that fused

  • one immortal and one bind ab

• separate and make clones to make mAb

  • we know exactly what the ab is

• ELISA

  • plate hope to have isolated clones of B cell

Agglutination reactions

• immune complex formation

• precipitation test to see what crosses

• immunoassay allows for this

• of blood cells

• ab, does it spread or is it concentrated to one area

• hemagglutination can be used to detect ag conjugated to surface RBC

Radioimmunoassay

• allows to measure very small amounts

• very sensitive

• relies on competitive binding of radio labeled or unlabeled to limiting amounts of ab

• ab - ag complexes can be sperated from unbound ag

• radiation counted

RIA

• measuring inhibition of binding

• make radioactive molecule to find

• some bound ab some not

• seperate bound and unbound

• radioactive ag and regular ag

• mix with same ab some will bind

• suppress amount of ab bound

• can detect small changes in radiation

ELISA

• assay can use ab and ag covalently bound to enzymes

• indirect

  • detect presence and concentration of ab in a sample

  • method of choice to detect presence of serum ab against HIV

  • ab constantly vary in amount of ag

western blot

• ag separated on gel

• separated proteins transfer to thin membrane

  • covalently linked

• membrane incubated with ab

  • ab bind to ag on membrane

• ab detected normally using emxymatic ab

  • anti Ig enzyme conjugates

  • one band = protein

anti HIV western

• useful in HIV gets protein to cross react with virus

• wither there will be many bands or only one

• when only one means that it cross reacted with the protein