Affinity purification of novel bispecific antibodies recognising carcinoembryonic antigen and doxorubicin.
We have developed a method which combines Protein A affinity chromatography and HPLC analytical and semipreparative hydroxyapatite affinity chromatography to purify bispecific antibodies (BsMabs) from hybrid-hybridomas secreting antibodies recognising carcinoembryonic antigen (CEA) and the chemotherapeutic drug doxorubicin (Dox). Elution of the HPLC hydroxyapatite columns with a 60-360 mM phosphate buffer gradient was found to give better separation than elution with a 60-180 mM phosphate buffer gradient. Careful monitoring of HPLC fractions by enzyme linked immunosorbent assays for anti-CEA, anti-Dox and dual anti-CEA/anti-Dox activity, and pooling of fractions on the basis of these results, enabled the purification of novel BsMabs for use in in vitro and preclinical in vivo experiments.
Ford
C H
CH
Department of Surgery, Faculty of Medicine, Kuwait University, Safat. ford@hsc.kuniv.edu.kw
Osborne
P O
PO
Mathew
A
A
Rego
B G
BG
D000818:Animals; D018033:Antibodies, Bispecific / Q000302:isolation & purification*; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods*; D004317:Doxorubicin / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D013205:Staphylococcal Protein A
D018033:Antibodies, Bispecific; D000970:Antineoplastic Agents; D002272:Carcinoembryonic Antigen; D013205:Staphylococcal Protein A; D004317:Doxorubicin