TECHNICAL PROBLEMS ARISING FROM THE USE OF THE IMMUNOBLOT FOR DETERMINATION OF THE REACTIVITY OF NATURAL ANTIBODIES WITH DIFFERENT LIPOPOLYSACCHARIDES (LPS)

Natural polyreactive antibodies (NPAB) appear to play an important rol e in the first-line defence against invading bacteria, The major const ituent of the outer membrane of Gram-negative bacteria is the lipopoly saccharide (LPS). Therefore, reactivity against this structure could b e of importance in protecting the organism from the harmful effects of LPS. Immunoblotting has become a common method to verify the specific ity of antigen antibody interactions. Various immunoblot techniques fo r testing the reactivity of monoclonal antibodies with LPS have been p ublished using nitrocellulose and detergent-free blocking buffer syste ms. These methods are not suitable for the investigation of NPABs due to the broad reactivity and a high background staining which gives ris e to interpretational difficulties. In the present study we demonstrat e an immunoblot technique using polyvinylidene difluoride (PVDF) membr anes and a detergent-containing buffer system which permits to detect LPS reactivity of NPABs. The polyreactive monoclonal human antibody CB O3 used was screened for lipid A/LPS reactivity in ELISA experiments. The binding was confirmed in the described blot system and depends on the membranes and blocking agents used. The use of nitrocellulose vers us PVDF was also tested for monospecific anti-LPS antibodies and the l atter can be recommended due to the production of stronger reaction pa tterns without any background staining.