title: A filtration method for rapid preparation of conjugates for immunoassay.
abstract: Modification of protein and other biopolymers by labeling them with small or macromolecules has become a very powerful research tool in biochemistry, molecular biology, diagnostics, and therapeutics. However, current methodologies available for their preparations are not straightforward and take several hours of incubation time. In this paper, we describe a new filtration-assisted technique for covalent conjugation between the reactive functional groups of two different molecules (small or macromolecules). Compared to the current method, this new approach significantly reduces the total reaction time from several hours to just a few minutes. The technique has been used for the preparation of conjugates of a small molecule to a protein such as biotin-BSA conjugate or small molecules to a small molecule such as biotin-tyramine conjugate or protein-protein conjugation such as antibody-horseradish peroxidase conjugate. The procedure consists of filtering the reaction mixture multiple times through membrane micropores with the help of two syringes, which make the cross filtration process less laborious. The method saves time, allows conjugation of less than 1mg protein and produces conjugates better than those obtained by the current methods. Although the present technique has been applied on some common conjugation methods, it provides a potentially general method, and may further be expanded for the synthesis of several other macromolecular conjugates.
authors.affiliation: Drug Development, Diagnostics and Biotechnology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Calcutta-700 032, India. tkdhar@iicb.res.in
authors.forename: Tarun K
authors.initials: TK
authors.lastname: Dhar
authors.forename: Subham
authors.initials: S
authors.lastname: Dasgupta
authors.forename: Dipika
authors.initials: D
authors.lastname: Ray
authors.forename: Meghna
authors.initials: M
authors.lastname: Banerjee
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001710:Biotin / Q000737:chemistry / Q000276:immunology; D002417:Cattle; D005374:Filtration / Q000379:methods*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000276:immunology; D013997:Time Factors; D014439:Tyramine / Q000737:chemistry / Q000276:immunology
chemical_list: D000906:Antibodies; D012710:Serum Albumin, Bovine; D001710:Biotin; D006735:Horseradish Peroxidase; D014439:Tyramine