Are there differences between using primary antibody (MAb) labelling kit and using conjugated 2nd Ab?
I usually use conjugated 2nd Ab with primary MAb to detect an antigen. However, I have heard about shorten way which is labelling primary MAb using a labelling kit such as Lightning-Link® Horseradish Peroxidase (HRP).
Many thanks in advance for your help.
The main differences are (i) fewer incubation and wash steps with direct labelling (ii) the secondary antibody is conjugated to HRP with the 2-step method, and in the shorter approach the primary antibody is covalently attached to HRP.
The Lightning-Link method is extremely simple and the only thing you need to do is make sure the antibody buffer is compatible with the conjugation reaction.
A lot of people assume that there is amplification with the two step method but this is often illusory because the primary antibody is dissociating in the secondary incubation and wash steps. Indeed for many antibodies that dissociate relatively quickly you may get even better results with direct labelling because there are fewer wash steps.
Dear Nick,
Really that is a fantastic explanation.
Do you think MAb buffer which is "Phosphate buffered saline pH7.2 with 0.095% (w/v) sodium azide" could affect on direct labelling?
Avoid using sodium azide in buffer. Prepare fresh buffer and use it. Yes, the use of primary antibody bound HRP will reduce assay time for detection of antigen.
Many thanks for your advice.
Again thanks for all who deposit an answer.
Marwan
Dear All,
I need to double check the procedure, please if you could help:
1. Before you add antibody to the Lightning-Link® mix, add 1µl of LL-modifier reagent for each 10µl of antibody to be labelled. Mix gently.
2. Remove the screw cap from the vial of Lightning Link® mix and pipette the antibody sample (with added LL-modifier) directly onto the lyophilized material. Resuspend gently by withdrawing and redispensing the liquid once or twice using a pipette.
3. Place the cap back on the vial and leave the vial standing for 3 hours at room temperature (20-25⁰C). Alternatively, and sometimes more conveniently, conjugations can be set up and left at room temperature overnight, as the longer incubation time has no negative effect on the conjugate.
4. After incubating for 3 hours (or more), add 1µl of LL quencher reagent for every 10µl of antibody used. The conjugate can be used after 30 minutes. No separation steps are necessary.
That means, that I have to develop reaction colour using TMB and then stop reaction with H2SO4.
Is there any comment regarding these steps please before I start my experiment?
Best Regards,
Marwan
- Badges
- Science topic