Development of a dot-blot assay for screening monoclonal antibodies to low-molecular-mass drugs.
Dot-blot is a versatile and simple analysis to perform. We adapted this method as a simple identity test for monoclonal antibodies to a number of small compounds: three transplant drugs, an anticonvulsant, a steroid, an anticancer drug, and an antibiotic. Immunology-based identity tests using low-molecular-mass organic compounds have historically been a challenge to develop. We modified the traditional dot-blot assay to serve as an identity test for monoclonal antibodies to carbamazepine, sirolimus, tacrolimus, cyclosporine, cortisol, methotrexate, and gentamicin. The primary obstacle was the immobilization of these organic compounds on nitrocellulose as nitrocellulose is also soluble in most of the organic solvents in which the compounds are soluble. We evaluated different membranes, solvents, and chemical forms of these organic compounds to overcome this challenge. A number of incubation and washing solutions were also investigated. By varying the chemical form, concentration, and incubation conditions, a set of effective and reproducible identity tests were developed for these monoclonal antibodies.
Rupprecht
Kevin R
KR
Diagnostics Analytical Chemistry Research, Abbott Laboratories, Abbott Park, IL 60064, USA. kevin.rupprecht@abbott.com
Nair
Rad K
RK
Harwick
Larissa C
LC
Grote
Jonathan
J
Beligere
Gangamani S
GS
Rege
Sushil D
SD
Chen
Yon-Yih
YY
Lin
Zhen
Z
Fishpaugh
Jeffrey R
JR
D000166:Acridines / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000276:immunology; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000927:Anticonvulsants / Q000737:chemistry / Q000276:immunology; D002220:Carbamazepine / Q000737:chemistry / Q000276:immunology; D003101:Collodion / Q000737:chemistry; D016572:Cyclosporine / Q000737:chemistry / Q000276:immunology; D005839:Gentamicins / Q000737:chemistry / Q000276:immunology; D006854:Hydrocortisone / Q000737:chemistry / Q000276:immunology; D015151:Immunoblotting / Q000379:methods*; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000276:immunology; D008727:Methotrexate / Q000737:chemistry / Q000276:immunology; D004364:Pharmaceutical Preparations / Q000032:analysis*; D015203:Reproducibility of Results; D020123:Sirolimus / Q000737:chemistry / Q000276:immunology; D016559:Tacrolimus / Q000737:chemistry / Q000276:immunology
D000166:Acridines; D000900:Anti-Bacterial Agents; D000893:Anti-Inflammatory Agents; D000911:Antibodies, Monoclonal; D000927:Anticonvulsants; D005839:Gentamicins; D007166:Immunosuppressive Agents; D004364:Pharmaceutical Preparations; C403604:acridinium I; D002220:Carbamazepine; D016572:Cyclosporine; D003101:Collodion; D020123:Sirolimus; D006854:Hydrocortisone; D016559:Tacrolimus; D008727:Methotrexate