Technical problems arising from the use of the immunoblot for determination of the reactivity of natural antibodies with different lipopolysaccharides (LPS)

Abstract

Natural polyreactive antibodies (NPAB) appear to play an important role in the first-line defence against invading bacteria. The major constituent of the outer membrane of Gram-negative bacteria is the lipopolysaccharide (LPS). Therefore, reactivity against this structure could be of importance in protecting the organism from the harmful effects of LPS. Immunoblotting has become a common method to verify the specificity of antigen antibody interactions. Various immunoblot techniques for testing the reactivity of monoclonal antibodies with LPS have been published using nitrocellulose and detergent-free blocking buffer systems. These methods are not suitable for the investigation of NPABs due to the broad reactivity and a high background staining which gives rise to interpretational difficulties. In the present study we demonstrate an immunoblot technique using polyvinylidene difluoride (PVDF) membranes and a detergent-containing buffer system which permits to detect LPS reactivity of NPABs. The polyreactive monoclonal human antibody CB03 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 versus PVDF was also tested for monospecific anti-LPS antibodies and the latter can be recommended due to the production of stronger reaction patterns without any background staining.