title: A detergent-based procedure for the preparation of IgG-like bispecific antibodies in high yield.
abstract: Bispecific antibodies (BsAbs), with the ability to recognize two different epitopes simultaneously, offer remarkable advantages in bioassays, cancer therapy, biosensors, and enzyme electrodes. Preparation and purification of BsAbs in adequate quantities remains a major hurdle in their use in various applications. Poor yield is also the principal limitation in the preparation of BsAbs by the redox procedure. IgG with reduced inter-heavy chain disulfides do not dissociate into half molecules at neutral pH. In this study, we report that the dissociation occurs in presence of sodium dodecyl sulphate (SDS) and inclusion of the detergent during the redox procedure results in remarkable increase in the formation of the BsAbs. Exposure of antibodies to 0.1% (w/v) SDS causes only minor loss in secondary/tertiary structure and the ability to bind the antigen. The BsAbs prepared using the modified redox procedure that recognize the antigens HRP and α-LA were prepared and successfully employed for detecting α-LA in milk/dairy products by ELISA and dot blot techniques. BsAbs were also prepared from partially purified immunoglobulin gamma (IgG). This work shows for the first time that SDS, by dissociating IgG with reduced inter-heavy chain disulfides into half molecules, markedly enhances the formation of BsAbs by the redox procedure.
authors.affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
authors.forename: Jyoti
authors.initials: J
authors.lastname: Gupta
authors.affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
authors.forename: Mehboob
authors.initials: M
authors.lastname: Hoque
authors.affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
authors.forename: Masihuz
authors.initials: M
authors.lastname: Zaman
authors.affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
authors.forename: Rizwan Hasan
authors.initials: RH
authors.lastname: Khan
authors.affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
authors.forename: M
authors.initials: M
authors.lastname: Saleemuddin
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D000067493:Dynamic Light Scattering; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007768:Lactalbumin / Q000032:analysis / Q000276:immunology; D008623:Mercaptoethanol / Q000737:chemistry; D008892:Milk / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D018033:Antibodies, Bispecific; D004220:Disulfides; D007074:Immunoglobulin G; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D007768:Lactalbumin; D006735:Horseradish Peroxidase