Why is the secondary antibody conjugated to the enzyme in ELISA, instead of the primary antibody? Wouldn't it be easier to conjugate the enzyme to the primary antibody?
Making an antibody-enzyme conjugate isn't trivial. By using a primary/secondary set-up you can use the same well-characterised conjugate in combination with many different primary antibodies (as long as these primaries are all raised in the same species). There is also the possibility of some amplification: for example, if the secondary is an anti-Fab then two secondary Igs will bind to each primary.
Response to OP comment
Most primary antibodies in common use are derived from rabbit or mouse, and most are IgG. So, for example, the secondary antibodies goat anti-rabbit IgG and goat anti-mouse IgG will cover most experiments.
Another reason that secondary antibodies are used is to look for the production of antibodies by a test animal. Follow me for a second with an example that I think will make it clear.
Our Purpose: We have identified a protein (call it 'V_Entry') that is vital for Ebola to infect human cells. We wonder if antibodies against V_Entry will block Ebola infection. We then use this V_Entry protein as a vaccine in a clinical trial.
Our Question: Do patients have generate their own anti-V_Entry antibodies as a response to the vaccine. (note: at this point we are not addressing whether these antibodies will be protective or not- that's a separate question)
Our basic protocol:
- Day 0: Take blood sample Vaccinate patients
- Day 14 Take Blood Sample Booster Patients
- Day 30 Take Blood Sample
To test the ability of the vaccine to generate an antibodies specific to V_Entry, we will assay serum from our cohort via ELISA.
Our Test: (I'm ignoring controls and other details here to be clear) Coat ELISA plates with V_Entry protein Add aliquots of serum from each of out patients at each timepoint. Wash Add a secondary antibody conjugated with a detection enzyme Wash Add substrate for Enzyme Allow Enzyme to develop the substrate (usually colorimetric) Read on a plate reader
Some expected results:
Nothing happens - a control well works, but none of our patients show antibody generation
Patients have pre-existing antibody that binds V_Entry (maybe these people were already exposed to Ebola?)
Patients have no pre-existing antibody, but they generate antibody in response to the vaccine (control patients not given vaccine show no new antibody)
Why Secondary Antibody was Necessary: We can't practically label every antibody made by the patient, but we can use a secondary antibody (perhaps Goat Anti-Human IgG) to detect any human IgG that has stuck to the V-Entry on our plate.
Was this a long run for a short slide? I considered making a slide for each step of the protocol, but I started wondering if I'm avoiding something...
protected by Community♦ Sep 15 '15 at 10:02
Thank you for your interest in this question.
Because it has attracted low-quality or spam answers that had to be removed, posting an answer now requires 10 reputation on this site (the association bonus does not count).
Would you like to answer one of these unanswered questions instead?