High efficiency immunoaffinity purification of anti-peptide antibodies on thiopropyl sepharose immunoadsorbants.
Antibodies to peptides are routinely made by immunizing animals with peptide linked to a carrier protein such as keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) via a disulfide bond. The majority of such a polyclonal antibody response is directed against the carrier protein. The presence of such background antibodies often complicates efforts to characterize the desired anti-peptide antibody; hence it is desirable to isolate the specific fraction of immunoglobulin reactive against the peptide of interest. We describe here a simple and efficient technique to purify anti-peptide antibodies from such sera using commercially available reagents. Peptide antigen with a carboxy or amino terminal cysteine is coupled to thiopropyl Sepharose via a disulfide linkage. The bond between peptide and propyl group on Sepharose was stable at neutral and acidic pHs, and affinity bound anti-peptide antibodies were eluted from the column at low pH (pH 3.0). This procedure permits purification of anti-peptide antibodies, separating them from usually high-titered antibodies to the carrier protein. We describe the application of this method for purification of antibodies to two peptides derived from the glycoprotein sequence of lymphocytic choriomeningitis virus, as well as sequences derived from the human acetylcholine receptor.
Parekh
B S
BS
Research Institute of Scripps Clinic, La Jolla, CA 92037.
Schwimmbeck
P W
PW
Buchmeier
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D007136:Immunoglobulins / Q000302:isolation & purification*; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits / Q000276:immunology; D011950:Receptors, Cholinergic / Q000032:analysis / Q000276:immunology; D012685:Sepharose / Q000031:analogs & derivatives; D014764:Viral Proteins / Q000276:immunology
D000906:Antibodies; D007136:Immunoglobulins; D010455:Peptides; D011950:Receptors, Cholinergic; D014764:Viral Proteins; C035872:thiopropyl-sepharose; D012685:Sepharose