2 types of Labelling
Monoclonal
Polyclonal
How are Antibody Labelling characterised by?
- Non covelent interaction (similar to the lock and key)
- Detection of Ag or Ab
- Diagnosis of disease
- Identify molecular biology and medical intrest
What is the basic concept of immunological assays?
Antigen A+Anti- Antigen A= Positive interaction
What celluar antigens can we target?
Cytoplasmic Nuclear Cell membrane Lipids Proteins
Why is labelled material usually used?
used to measure the amount of antigen or antibody present
3 labelling methods?
- Immunoenzyme methods
- Immunofloresence methods
- Immuofold methods
description of the immunoenzyme method?
Enzyme used as a label
eg, Peroxidase, Glucose Oxidase
description of the immunofloresence method?
Flourochrome used as a label
e,g AMCA, Flourscin, TRITC
Immunogold method description?
Collodial gold particles are used as the label
USed in microscopy
Direct labelling methods?
Label attached directly to the primary antibody. Used in kidney biopses
Indirect labelling methods?
Label attached to a secondary antibody:
- allows greater amplification
- More economic
- Longer
5 Types of antibody load?
- Enzymes
- Biotin
- Flourophores
- Gold
- Radioisotope labels
Enzymes acting as an antibody load description?
2 typical substances:
- 5-brono-4-chloro-3’indolyphosphate ptolyphosphate salt (BCIP)
- Nitro blue tetracolium chloride (NBT)
Preparation for enzyme conjugates?
Chemical cross linking
Size exclusion chromatography
Biotin description?
Covelently linked to Fc domain using aminocapric acid
Free biotin removed by gel filtration
Optimally 20-30 biotin
Amplification step
What is the signal amplification with biotin labelling?
Label attached to a secondary antibody:
-high sensitivity
-Longer + more expensive
greater signal amplification
Fluorophores description
Excitable molecules with distinct excitation/emission wavelengths–> eg, FITC
What can a Flow Cytometer tell us about a cell?
Its relative size
Its gravity
Relative flouresence intensity
Gold description?
Immunoglobulin gold complexes
Antibody + collodial gold + PEG (polyethylene Glycol)
Centrifuge conjugates on sucrose density gradient
Radioisotope labels advantages?
Flexibility
Size
Sensitivity
Radioisotope label disadvantages?
Toxicity
Shelf life
