A method for the detection of serologically crossreacting antigens both within and between protein mixtures: the western cross blot.
The method described in the present paper permits the detection of antigenically related proteins within or between different antigen mixtures. For this purpose two separate SDS-polyacrylamide gels are run (the antigen mixture is applied to the entire width of the gel) and each gel is blotted on to a separate nitrocellulose paper. One sheet ('donor sheet') is incubated with antiserum and placed on to the other blot ('receptor sheet') so that the antigen bands are perpendicular to each other. Subsequently the antibodies from the donor sheet are blotted on to the receptor sheet in alkaline borate buffer containing 1 M NaSCN, which dissociates antigen-antibody complexes. After the removal of the donor sheet the receptor sheet containing the transblotted antibodies is equilibrated in phosphate-buffered saline in order to reconstitute the binding conditions. Antibodies which have bound to receptor antigens during this equilibration period are detected by the use of a peroxidase labelled 2nd antibody. Because each band on the donor sheet crosses each band on the receptor sheet during the transfer, the antibodies from each band of the donor sheet are able to react with each antigen band of the receptor sheet. The validity of the method was established by demonstrating the crossreactivity of a polyclonal antiserum against the intact bovine gamma globulin molecule with the heavy and light chain subunits (and partial reduction products of the molecule) and the absence of crossreactivity between heavy and light chains. In a second experiment crossreacting antigens of different molecular weight were detected within several strains of Escherichia coli. In addition, comparisons of different strains of Escherichia coli revealed crossreacting antigens of identical as well as of different molecular weight.
Hammerl
P
P
University of Salzburg, Department of Biochemistry, Austria.
Hartl
A
A
Freund
J
J
Thalhamer
J
J
D015153:Blotting, Western / Q000379:methods*; D003429:Cross Reactions; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000276:immunology*; D012995:Solubility; D013861:Thiocyanates
D011506:Proteins; D013861:Thiocyanates