A novel affinity purification method to isolate peptide specific antibodies
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A novel affinity purification method to isolate peptide specific antibodies. / Karlsen, Alan E; Lernmark, A; Kofod, Hans; Dyrberg, T.
In: Journal of Immunological Methods, Vol. 128, No. 2, 17.04.1990, p. 151-7.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - A novel affinity purification method to isolate peptide specific antibodies
AU - Karlsen, Alan E
AU - Lernmark, A
AU - Kofod, Hans
AU - Dyrberg, T
PY - 1990/4/17
Y1 - 1990/4/17
N2 - Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displaced by the immunizing but not by unrelated peptides.
AB - Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displaced by the immunizing but not by unrelated peptides.
KW - Animals
KW - Antibodies
KW - Antibody Specificity
KW - Chromatography, Affinity
KW - Electrophoresis, Polyacrylamide Gel
KW - Enzyme-Linked Immunosorbent Assay
KW - HLA-DQ Antigens
KW - Immunoblotting
KW - Peptides
KW - Rabbits
KW - Rats
KW - Receptors, Somatotropin
M3 - Journal article
C2 - 2324508
VL - 128
SP - 151
EP - 157
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 2
ER -
ID: 45574812