Before performing the labelling, several parameters should be optimised. Adverse effects can indeed be observed on the antibodies immunoreactivity. This first labelling must be considered as a feasibility study. To determine the best coupling conditions, each project includes the following steps :
- conjugation of antibodies with a fluorochrome or biotin at three different concentrations
- conjugation of antibodies with an enzyme at one concentration (according to the literature)
- control of the antibodies immunoreactivity before and after conjugation (the immunogen is required to perform this step, please for more details)
- shipment of conjugated products samples to your laboratory so that you can determine the dye/antibody ratio which yields the best signal.
According to the nature of the dye, different methods are carried out:
- fluorochromes and biotin are coupled to 100 μg of antibody via an activation by isothiocyanate or a succinimidyl ester, respectively
- ALP is coupled to 100 μg of antibody using the technique set up by Avrameas et al.
- HRP is coupled to 100 μg of antibody using the technique set up by Nakane et al.
Labelled antibodies are provided in a phosphate buffer saline containing BSA, thimerosal or sodium azide, glycerol 50%.
Following the feasibility study and upon your confirmation according to the results of your tests, the optimal dye/ antibody ratio will be used to produce conjugated antibodies in higher amount.
- estimation of the molar ratio (for fluorochrome- and biotin-conjugated antibodies),
- control of the immunoreactivity of the antibodies after labelling (the immunogen is required to perform this step, please for more details),
- study of the enzymatic activity (for enzyme-conjugated antibodies).
This service is performed for a minimal quantity of 500 μg of antibody.