Antibody techniques Flashcards
Antigen
Substance that binds to Ab or T cell receptor
what part of Ag does Ab recognise ?
Epitopes
Ab structure
4 polypeptide chains
Variable region: ABS
constant region
Polyclonal
Mixture of Abs recognising Several epitopes of 1 Ag
Monoclonal
1 clone Of Ab recognises 1 epitopes = highly specific
polyclonal in serum come from?
activation of many B cells with specificity for diff epitopes on Ag
Monoclonal come from
clones of individual B cells + have specificity to 1 epitope
Monoclonal production Method
1) Animal Immunized with Ag
2) B cells removed from spleen
3) B cells + myeloma cells fuse = hybridoma
4) Cultured on HAT= only hybridoma grows
5) screen for specific Ab production
6) Desired Ab grown
7) Ab Isolated + purifed
why myeloma cells?
cancer cell that grows continuously
What HAT medium does
only hybridoma grows
unfused dies
Hybridoma
B cell + myeloma cell fused together
Screening
separated in microtiter plate t tested ability to produce specific Ab
Polyclonal pros v cons
inexpensive, quicker
less specific, Batch variability
monoclonal pros v cons
Expensive, longer, may be too specific
more specific, All batches identical
Lab uses of mAb(x3)
Detect analyte in tissue (IHC)
Derect analyte in liquid (ELISA)
separation of analyte from mixture (immuno precipitation)
what Immunoassay uses :
Detectable label attached to Ab E.g fluorescence, enzyme, Radioisotope
what immunoassay does?
Quantify soluble Ag
Non - competitive immumoassay Example
sandwich ELISA
sandwich ELISA method
1) Well coated with capture Ab
2) sample Ag binds Ab
3) 2nd detection Ab with enzyme binds Ag
4) add substrate = colour change
Sandwich ELISA detection
Spectroscopy
HRP -linked AB
Binds detection Ab= amplified signal
Competitive Immunoassay difference
Analyte competes for Binding site
How Non / competitive measured
Non= occupied sites of labelled Ab
competitive= unoccupied sites
competitive method
1) Sample + labelled Ag added to capture Ab
2) Either Ag or sample binds
3) Substrate added= colour change
4) measures Ab unbound with Ag
Non competitive graph
directly proportional : increase conc= Increase absorbance
competitive graph
inverse correlation : more signal less Ag in sample
immunoassay to measures ab + Example
measure levels of Ab by coating wells with Ag
E. g diagnose Coeliac
Diagnosis of Cs
ELISA detects patients Ab against tissue Transglutaminase+ Gladin
IHC benefits (x3)
Qualitative, Gives location + identify cell events
ICC method
1) Tissue / cells on slide
2) Primary Ab binds
3) wash unbound
4) Secondary labelled Ab binds
5) wash unbound
6) substrate colour change
Signal amplification : Biotinylated Ab
Avidin: 4 bunding sites bind Biotin
Biotin: coenzyme binds Protein
IHC / ICC example diagnosis
ANA detects auto antibodies using fluorophore Linked Ab =Specific staining pattern
Biotinylation
using Biotin+ Avidin to amplify signal
