A new approach of endotoxic testing by using a monoclonal antibody against endotoxin (WN1-222/5) and flow cytometry

Serum-endotoxin was formerly measured by the limulus-amebocyte assay. A major step forward since this test assay was available was the introduction of chromogenic substrates which were convertible by LAL clotting enzymes. Further improvements could not prevent the results to be non-specific and in general unsatisfactory. An alternative approach of our group for the detection of endotoxin was the usage of a cross reactive monoclonal antibody against endotoxin (WN1-222/5) in combination with flow cytometry, measuring subsequent light emission of a second antibody directed against WN1-222-5 in peripheral mononuclear cells (MØs).

In our porcine endotoxin shock model we investigated 10 pigs under analgosedation receiving 250 ng/kg/hour endotoxin from Salmonella friedeman.

Separation of mononuclear cells every 4 h and determination of the concentration of endotoxin revealed the results shown in the Table.

Table 1

In this preliminary study we could not find LPS at the cell surface in vivo but in vitro (unpublished data). The current results indicate that the process underlying LPS internalization are very complex. During the course of our endotoxin shock with continuous endotoxin infusion endotoxin internalization increases gradually. Initially the PMNs show the highest activity and a small increase thereafter. In contrast the MØs revealed a more than 10 times higher activity after 4 hours of the experiment.

With our new specific endotoxin test protocol it might well be possible in the future to evaluate the different responses of LPS as it finds its way to different surface domains or intracellular components in different cell populations, respectively.