• flow cytometry• gel electrophoresis• immunoprecipitation (IP)• immunoblotting

• microscopy• immunofluorescence (IFA)• electron microscopy

• ELISA

Analytical Techniques Utilizing Antibodies

ImmunofluorescenceUsed to:• localize antigens to specific cells or

subcellular structures• detect and quantify antibody

General Procedure:• incubate cells or tissue with antibody• detect Ag-Ab complex with conjugated

secondary antibody• fluorescence (examine under UV

illumination)• enzyme (substrate forms precipitate)

IFA Protocol

1. Prepare cells or tissue.

2. Incubate 1o antibody.

3. Wash.4. Incubate 2o

antibody.5. Wash.6. View under UV

illumination.

Preparation of Cells• section and mount tissues• grow adherent cells on micro-

scope slides or cover slips• affix suspension cells on

microscope slides• carry out incubations in

suspension• ± fixation • organic solvents• paraformaldehyde• <0.1% glutaraldehyde

• ± permeabilization (eg., detergents)

• surface labeling of unfixed cells

epifluorescence bright field

•bright image against dark background•corresponds to location of antigen

+ 0.1% TX-100

Detergent Permeabilization

EtBr stains DNA and RNA

DAPI stains only DNA

Counter Staining with Fluorescent

Dyes

Dual-Labeling Experiments

•determine extent of co-localization•use 1o antibodies from different species and 2o antibodies labeled with different fluorochromes

•label 1o antibodies with different fluorochromes

Immuno-Electron Microscopy

• prepare samples• optimize fixation conditions• use resins that polymerize at RT

• ‘float’ grids on drops (1o and 2o abs, washes)• surface of section accessible to

antibodies (± 'etching')• 2o-Ab conjugated with colloidal gold• size ranges from 5-15 nm• enzyme linked (electron dense precipitate)

Ultrastructure vs. Labeling

• fixation conditions preserving ultrastructure lead to loss of labeling

• cryo-electron microscopy• special microtome and

stage

AccessibilityProblems

• ultrasmall gold (<1 nm)• + silver

enhancement• ±pre-embedding

• Use same antigen with different antibodies• Quantify by serial dilutions

Characterizing Antibodies

IFA

• bind antigen to 96-well microplate (or membrane)• neg. (and pos.) controls• purity?

• incubate with 1o and 2o antibodies• use soluble chromogenic

substrates in 96-well plates• quantify Ab or Ag

Conventional ELISA

• measure absorbance with ELISA microplate reader

ELISA Variations

• radio-immunosorbent assay (RIA)

Generic Immunoassay Procedure•form antibody-antigen complex•detect Ag-Ab complex•labeled anti-antibody (2o Ab)•labeled protein A or G•directly label 1o Ab

Direct vs.

Indirect

less stepsless bkg (?)dual label

convenienceamplification (?)

Fluorochromes•fluorescein• rhodamineEnzyme

Crosslinking•AP•HRP Radiolabeling• iodination•metabolically

(mAbs)Biotinylation

Biotin-Avidin Detection Systems

•label 1o- or 2o-Ab with biotin

•detect with avidin labeled with marker

•high affinity may increase sensitivity

•more steps

TECHNIQUEGENERAL PROCEDURE

TYPICAL APPLICATION

ELISA

adsorb Ag to solid support

incubate with Ab detect bound Ab

quantify Ab quantify Ag process large # of

samples

BLOTTING

SDS-PAGE and transfer

incubate with Ab detect bound Ab

identify subunit MW quantify Ag quantify Ab?

IFA fix cells on slide incubate with Ab detect bound Ab

subcellular localization quantify Ab quantify Ag?