Isolation, characterization and comparison of antipeptide and antiprotein rabbit antibodies to the pi-isoform of glutathione S-transferase.
The main linear epitopes of pi-glutathione transferase (pi-GST, EC 2.5.1.18), an enzyme related to cancer progression in a restricted number of tumours, were identified by testing in ELISA the reactivities of polyclonal anti-pi-GST rabbit sera against a panel of 51 overlapping decapeptides, covering the whole 216-residue sequence of the protein. Several major reactivity peaks were detected, each covering two or three adjacent peptides. The most active fragments were then reconstructed by conventional solid-phase synthesis, linked to Sepharose, and used as affinity ligands for isolating specific anti-pi-GST antibody subsets. A second group of antisera was then prepared in rabbits by using as immunogens some of the above described synthetic fragments, linked to a carrier protein, and antipeptide antibodies purified by affinity chromatography. An ELISA test was then performed, using as antigens a panel of peptides and different isoforms of GST, in order to establish whether antibodies isolated from total anti-pi-GST sera would display higher reactivity and specificity, as compared to traditional antipeptide antibodies. Binding data clearly confirm that the formers might be indeed better reagents for the detection and possibly quantitation of pi-GST.
Di Modugno
F
F
Laboratory of Biochemistry, Regina Elena Institute for Cancer Research, Roma, Italy.
Rosano L
Castelli
M
M
Chersi
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005982:Glutathione Transferase / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011817:Rabbits
D000906:Antibodies; D000939:Epitopes; D007527:Isoenzymes; D010446:Peptide Fragments; D005982:Glutathione Transferase