title: Immunoblot method for identifying surface components, determining their cross-reactivity, and investigating cell topology: results with Haemophilus influenzae type b.
abstract: Methods currently used for identifying exposed membrane components of gram-negative bacteria can give false positive results, and also do not provide information on nonprotein components such as lipopolysaccharide and polysaccharide capsules. A method, described within, has been developed to overcome these limitations. Briefly an outer membrane preparation derived from encapsulated (type b) Haemophilus influenzae was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated components were then transferred electrophoretically to nitrocellulose. The nitrocellulose was cut into vertical strips, which were then each incubated with rabbit antiserum to the whole bacterium or with the same antiserum after absorption with any of the following: the same strain of H. influenzae, a capsule-deficient mutant of that strain, other strains of H. influenzae, or other bacteria. The strips were then incubated with 125I-protein A, and the bound antibodies were detected by autoradiography. The autoradiograph of the strip exposed to unabsorbed antisera revealed the identity of those individual outer membrane components that bound antibodies. A comparison of the intensity of the various bands on this strip with those on the strips exposed to absorbed antisera was then used to identify (1) surface-exposed components, (2) those components occluded by capsule, and (3) cross-reactivity of exposed components. This method should be applicable to other cells and subcellular particles. Its major disadvantage is that it can provide false negative results.
mesh_terms: D001345:Autoradiography; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D003101:Collodion; D003429:Cross Reactions; D003720:Densitometry; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006193:Haemophilus influenzae / Q000032:analysis* / Q000648:ultrastructure; D007120:Immunochemistry; D013194:Staining and Labeling
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003101:Collodion