Systematic fractionation of serum antibodies using multiple antigen homologous peptides as affinity ligands.
The fractionation of polyclonal antibodies on multiple peptide ligands is described. The method is an application of a procedure for the synthesis of large numbers of peptides on individual polyethylene pins (Geysen et al., 1987). In this application, each pin-bound peptide is used as an affinity support. Antibodies bound to the peptides are then eluted, using buffers of either high or low pH. Each eluted antibody is then tested for specific binding to peptides or proteins, using ELISA procedures. A rabbit antiserum raised to gonococcal pilin was fractionated on a complete set of octapeptides homologous with the sequence of the pilin protein. Antibodies eluted from some of the peptides bound to pilin in solution. In a second example three hyperimmune sera raised to three different potyviruses were fractionated on their respective homologous peptide sequences. Testing the eluted antibodies on the three virus coat proteins revealed peptides which bound cross-reacting antibodies. Thus the method can be used to confirm direct peptide binding evidence for sequential epitopes. These peptides can then be used in affinity chromatography to increase the specificity of polyclonal sera. This can be achieved either by elution of the specific antibody from the peptide or by removal of cross-reacting antibodies from the whole serum by absorption on peptide.
Tribbick
G
G
Coselco Mimotopes Pty. Ltd., Clayton, Victoria, Australia.
Triantafyllou
B
B
Lauricella
R
R
Rodda
S J
SJ
Mason
T J
TJ
Geysen
H M
HM
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000302:isolation & purification*; D000914:Antibodies, Viral / Q000302:isolation & purification*; D000918:Antibody Specificity; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D015153:Blotting, Western; D002213:Capsid / Q000276:immunology; D002846:Chromatography, Affinity; D036781:Fimbriae Proteins; D006422:Hemerythrin / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000276:immunology; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology*; D010942:Plant Viruses / Q000276:immunology; D011095:Polyethylenes / Q000737:chemistry
D000907:Antibodies, Bacterial; D000914:Antibodies, Viral; D001425:Bacterial Outer Membrane Proteins; D006422:Hemerythrin; D008024:Ligands; D009842:Oligopeptides; D011095:Polyethylenes; D036781:Fimbriae Proteins