Development and validation of an indirect enzyme-linked immunosorbent assay (ELISA) for the nonsteroidal anti-inflammatory drug S-ibuprofen.
An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the nonsteroidal anti-inflammatory drug (NSAID) S-ibuprofen. Conjugates for immunization were prepared by linking S-ibuprofen via the spacer 4-aminobutyric acid to bovine serum albumin as well as to a novel synthetic lipopeptide using the N-hydroxysuccinimide/dicyclohexylcarbodiimide method. Immunization with these immunogens was carried out in New Zealand rabbits. A poly-L-lysine-S-ibuprofen conjugate was used as a hapten-carrier for coating the surface of the microtiter plates with the hapten. Horse-radish peroxidase labeled anti-rabbit IgG served as secondary antibody using hydrogen peroxide and ABTS as substrates. The characterization of the polyclonal antiserum with compounds of analogous structure demonstrated that the antiserum possesses a very high specificity for S-ibuprofen (cross-reactivity < 0.14-1.4%). Additional cross-reactivity experiments using R-ibuprofen (cross-reactivity 50.5%), ibufenac (58%) and isopropylphenylacetic acid (6.4%) were carried out to obtain more detailed information about the antigenic recognition concerning the chiral center. The results indicated that the polyclonal antiserum possesses an additional antibody population, whose antigenic recognition did not contain the chiral center. The upper and lower limits of quantification of the developed ELISA were defined as 362 and 3.62 ng S-ibuprofen/ml, respectively, based on a 90% confidence interval.
Gräfe
K A
KA
Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Frankfurt, Germany. K.Graefe@pharmchem.uni-frankfurt.de
Hoffmann
H
H
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000737:chemistry; D007052:Ibuprofen / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011817:Rabbits / Q000276:immunology; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry; D005719:gamma-Globulins / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D006241:Haptens; D005719:gamma-Globulins; D012710:Serum Albumin, Bovine; D007052:Ibuprofen