I have been trying to develop an ELISA assay to identify the presence of the whole parasite in culture by targeting one of its membrane protein. I recently found out that there is no signal difference between a culture medium containing my target cells and a culture medium free of my target cells, I used the medium as a control and it gave a high background (the medium composition RPMI, hypoxanthine,gentamycin an A+ human serum). I am suspecting, and my data suggests, that there might be a cross-reactivity between my antisera and the human serum, but I still can't figure out why. Any explanations?
Could it be that this cross reaction will prevent my antibody from reacting with the target protein on the surface of the parasites?
Thank you Seppe, for the input. I failed to precise that it is malaria parasite and it is cultivated in vitro using human blood and serum since the parasite live in the red blood cells. When I used albumax (which is an artificial serum) or PBS , there is no background. I would definitely check another batch of medium (serum).
Our blood donors are living in Germany, which is non endemic for malaria, it would be very surprising to have a contaminated serum.
Thanks once again.
Thank you Seppe, I would check that first and would let you know the outcome. Many regards.
Hi Aminake. Do you know if the blood you are using is single donor blood or pooled blood? If it is pooled blood then there is higher probability of having a contribution from someone who has been exposed to malaria.
Hi Steingrimur, I am pretty sure it might come from multiple donors, but I would double check. However I have confirmed that the Goat anti-mouse antibody do react with the medium supplemented with A+ serum as predicted by Seppe. While no reaction was observed in the non-supplemented medium.
Did you use a biotinylated secondary antibody? Is it possible that your hypoxanthine is binding to biotin?
Hi Balamayooran, I am not using biotinylated secondary antibody. Detecting the whole organism seems to be challenge in our case since we don't know how epitopes are presented , When we lyse the whole culture we are able to have a better detection pattern.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins