Hi Anya.

There are different methods to desorptions antigens from AlOH but in general have a poor efficiency. Hoewer, for your purpose, the most important is that, in your final sample to coat the microplate, there isn´t absolutly nothing of AlOH, because this compound join proteins and your conjugates will be captured by the AlOH coated and always your ELISA run wrong.

I suggest you the citrate method. Mix 1g of Sodium citrate/10 ml of vaccine. Shake ON at 37ºC and centrifuge to obtain the supernatant. Filter this supernatant by 0.45 µm (this is very important to eliminate all rest of AlOH), and finally concentrate between 20-100 fold (because normaly the vaccines have not high amount of antigens ) In this final sample you must have sufficient antigen for your ELISA

Thanks for the response. Do you know anything about Quil-A Saponin adjuvants? Whether they interfere in an ELISA and how to separate the antigens from them?

Also, how do you recommend I concentrate the antigen at the end of the desorption?

Hi Anya. The Quil A Saponin adjuvants have AlOH and saponine, but the saponine doesn´t interfere in ELISA. You can separate the AlOH and antigen by citrate method.

To concentrate the antigen at the end of the desorption I use concentratrors by centrifugation (Vivaspin 20 of Sartorius or Ultracell of Millipore are good)

Saponins are detergents and hence can interfere with ELISA, especially with antigen adsorption onto ELISA plates. To get rid of saponins, look for a system of their selective extraction. If antigens are peptides with free C-terminal carboxyl and N-terminal amino-groups, certain organic solvents with enhancing salt concentration of water layer may help. It's a theoretical consideration solely, I had no experience of that kind.

I carried out the AlOH desorption and there is definitely more protein in the supernatant then the pellet according to ELISA and a Bradford assay. I concentrated the antigens in the supernatant by centrifugation using a 0.2um filter (Vivaspin 20), however now I am seeing proteins in both the concentrate and the filtrate (according to SDS-PAGE). Does AlOH (if there is any remaining in the supernatant) interfere with SDS-PAGE? Do you think a 0.2um filter is too big? Also, does anyone know how a "0.2um" filter converts into MWCO (kDa)?

I seem to have removed any inference from the Quil-A saponin in my other vaccine with ultrafiltration. Thanks for your advice!

Hi Anya,

0.2 mkm filter is not good for concentrating proteins, it's for separating particles larger than 200 nm. This filter is good for sterilization of solutions and cleaning of particulates. To concentrate proteins by ultrafiltration, you need to use ultrafiltration devices with smaller pores. They are usually marked according to molecular masses of globular proteins/peptides they can retard - 5,000 Da, 10,000 Da and so on. Choose the one that definitely retards your antigens.

The fact that some amount of your proteins are retarded by the 0.2 mkm filter may mean that 1) they form aggregates larger than 0.2 mkm, and you have a colloidal solution of such aggregates, which also may be in an equilibrium with mono- or oligomers of the proteins; during the ultrafiltration you separate these fractions; 2) Al(OH)3 is still present in your solution in a certain amount, and forms colloidal particles that adsorb antigens on their surface and are retarded in the concentrate during the ultrafiltration.

A good qualitative reaction on Al+++ with alizarin exists. I have a procedure description, but it is in Russian.

Regerding the removal of saponin by ultrafiltration. It means that saponin has rather high critical micellar concentration and does not form tightly bound complexes with antigens. It's a good information for me. By the way, how many cycles of ultrafiltration have you performed for removing the Quil-A saponin interference in ELISA?

Hi Anya,

Your question is somewhat not clear. If you do not know the exact composition of the proprietary formulation how can you be able to detect these antigens after desorption?

Which antibodies did you like to use against these multivalent antigens?

Why you want to coat the microplates with multivalent preparation instead of monovalent antigens since you do not know the exact composition and how can you make sure the binding of multivalent preparations?

Desorption with sodium citrate won’t work for Al(OH)3 based vaccines for 100% desorption, as said by someone in this forum.