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For the TGF beta ELISA assay, what is the rationale for treating cell culture supernatants with 1 N HCl before assaying? The protocol states that it is to "To activate latent TGF beta 1 to the immunoreactive form". Does that mean the epitopes that the antibodies recognize will not be available without this treatment?
Asked on Nov 20 2012
The laboratory that developed the ELISA did not send data showing results +/- acid treatment but did send the following summary. Please let me know if you still have questions or concerns.
It is strongly recommended that the customer performs the acid extraction as free/active TGF-beta 1 is likely innately very low in abundance in most normal samples. If however the customer’s samples are somehow treated and/or diseased with expected elevated levels of active TGF-beta 1, then she would not necessarily have to. In brief:
No Extraction – the kit will only detect the innately active TGF-beta 1. (i.e. TGF-beta 1 which is already free, not bound). The bound latent complex form will not be detected.
Extraction – the kit will detect both the innately active TGF-beta 1 and the now released active TGF-beta 1 (so total TGF-beta 1).
According to our data on normal human serum samples, the apparent concentration of TGF beta 1 without activation is around 100 pg/ml. The OD value was pretty low with a 2-fold dilution – close to background. After extraction, we got around 2000 pg/ml. So there is a significant difference.
Abcam Scientific Support
Answered on Nov 20 2012