[Further development of a cell culture model for the detection of bacterial pyrogens]

In many cases the limulus amoebocyte lysate assay (LAL) is now accepted to detect pyrogenic contaminations in parenteralia instead of the rabbit pyrogenicity test. As the LAL test is able to detect only compounds of gramnegative bacteria (endotoxins) and as it is difficult to test samples with high amounts of protein, we have now further developed our previously established in vitro assay for the detection of bacterial pyrogens in order to overcome the shortcomings of the LAL assay. Our test system is based on the stimulation of IFN-gamma-primed human (THP-1) and murine (J774A.1 and RAW 264.7) monocytic cell lines by bacterial pyrogens to form neopterin or nitrite, respectively. THP-1 in serum-containing media can be used as a high sensitivity assay, while RAW 264.7 in serum-free media is a very good screening system. We showed that the cell-free supernatants of various S. aureus cultures could not be assessed by the LAL assay while the here presented cell culture model detected them very sensitively. The stimulation of immune cells seems to be caused by substances of various molecular masses. In conclusion, we suggest to reduce or even replace the rabbit pyrogenicity test by using our cell culture model in addition to the LAL assay.