Antibodies as biochemical reagents Flashcards
What is required for a successful immunoassay?
High affinity and specificity
Why use indirect immunoassay with a conjugated secondary antibody?
Indirect immunoassays are better as secondary antibody amplifies the signal. Also difficult and expensive to make every tagged antibody specific to the antigen.
What are capture immunoassays (example of ELISA)?
First Ab immobilized on plastic multi-well plate. Sample containing Ag applied, Ag binds, other components washed off. Secondary Ab applied. Binds to Ag on different epitopes. Labelled Ab binds to second anti-Ag specific Ab. Washed.
What does ELISA?
Enzyme linked immunosorbent assays.
What are some common enzymes used in ELISA?
Alkaline phosphatase (AP), horseradish peroxidase (HRP).
How are ELISA results read?
Read in microtitre plate reader so requires a standard curve to quantitate antigen which is the top row.
What happens in competitive immunoassays?
Capture antibody immobilised. Antigen mixed with labelled antigen and added to immobilised Ab. Labelled and unlabelled antigen bound by capture Ab. The higher the concentration of antigen in sample the less labelled antigen is captured. The lower the resulting signal.
What is enzyme multiplies immunoassay?
Quantitate small molecules such as drugs in blood or urine. Sample containing antigen, antibody and enzyme-linked antigen. Compete. When enzyme linked antigen is bound to antibody it is inactive. If it remains unlinked to antibody it converts the substrate into a detectable product showing colour change. More antigen in the sample the greater the signal.
What is a lateral flow?
Dipstick assay. Covid or pregnancy. Sample containing antigen mixed with labelled antibody e.g., gold particle. Sample +Ab pulled across device by capillary action. Meets line of immobilised capture Ab (anti-antigen). If antigen present immune complex of capture Ab-antigen labelled Ab forms and coloured line appears. Labelled Ab without bound analyte drawn across second line of immobilised Ab as a control line.
What is western blotting/ immunoblotting?
Protein solubilised in denaturing buffer containing ionic/charged detergent such as SDS. Resolved on basis of size and charge. Proteins transferred to membrane e.g., nitrocellulose PVDF immunoblots. Abs bind to immobilised Ag on membrane. similar to indirect non competitive immunoassay. More for research not clinical use.
What is the process of immunofluorescence/immunohistochemistry?
Permeabilise tissue/cell, incubate with labelled antibodies, use microscope to reveal distribution abundance of antigen. Multiplexing, super-resolution and immunoelectron microscopy allow precise localisation 100nm.
What does FISH do?
Detect labelled nucleic acids.
What is immunoprecipitation?
Analysis of protein: protein interactions etc. Antibody-protein A/G-bead conjugate (bacterial Ab binding proteins). Mix with sample, pellet beads, wash off unbound material. ID precipitated material using western blotting, mass spec.
What is ChIP-seq?
Chromatin immunoprecipitation + sequencing
What are the applications of ChIP-seq?
DNA-protein interaction, gene expression/regulation- nucleosome mapping, Epigenome mapping- post translational modification of histone.
