Over the past decade, phage display has maturated to be a frequently used method for the generation of monoclonal antibodies of human origin. The essential step of this method is the "biopanning" of phage carrying functional antibody fragments on their surface on an immobilized antigen. The screening of large combinatorial gene libraries with this method usually leads to a set of diverse clones specifically binding to the antigen that need to be characterized further. Beside its specificity, the key parameter to be determined is the affinity of the recombinant antibody fragment to its antigen. Here, we present a mass sensitive microsensor method that allows the estimation of antibody affinity directly from the phage supernatant. Binding of phage antibodies to the antigen immobilized on a quartz crystal microbalance (QCM) induced a mass dependent decrease in frequency. This principle was used to determine the apparent affinity of a single-chain (sc)Fv antibody against the RNA polymerase of Drosophila melanogaster presented on the surface of a filamentous phage (M13) from its association and dissociation rates. The apparent affinity obtained is in accordance with the affinity of the scFv fragment as determined by conventional equilibrium enzyme-linked immunosorbent assay (ELISA) and plasmon resonance methods.
Ring core-biotinylated testosterone tracers were synthesized with bridges of three different lengths connecting the biotin moiety to the steroid core (7α-Cn-Bio-T, n = 3, 6, or 11). Together with a position 7-specific polyclonal anti-testosterone antibody, we used the 7α-C11-Bio-T tracer to develop a novel, labeled-hapten competitive immunoassay for total testosterone in serum. (The C3 and C6 tracers proved to be not suitable for analogous immunoassays.) Enhanced chemiluminescence signal was generated by use of a second immobilized antibody and a streptavidin–horseradish peroxidase conjugate. The measuring range of the assay is 0.2–20.0 nmol/L, linearity of serial dilutions can be demonstrated, the lower detection limit is 0.125 nmol/L, and the interassay imprecisions are 13–16%. Accuracy determinations in mass spectrometry-controlled reference specimens showed a mean recovery of 95%. In addition, the assay shows low cross-reactivities, demonstrating the favorable specificity of the combination of a “nearly native” tracer with a position analog antibody. The optimized steric structure and the long spacer arm of the biotinylated testosterone tracer make this chemiluminescence assay well-suited for measuring total testosterone concentration in serum.
We describe the development and validation of a labeled-hapten competitive immunoassay for determining total estrone in serum. For the hapten tracer we use the 6 alpha-biotinylated estrone derivative, 3-hydroxyestra-1,3,5(10)-trien-17-one 6 alpha-N-(epsilon-biotinyl)aminocaproamide (Bio-E1). A specific polyclonal rabbit anti-estrone antibody is indirectly bound via an immobilized donkey anti-rabbit antibody on microtiter plate wells. The amount of Bio-E1 bound is then measured with streptavidin-horseradish peroxidase conjugate, whereby the enzyme activity is quantified by an enhanced chemiluminometric method. For the assay, serum samples were extracted with solid-phase extraction cartridges. The assay dynamic range was 93-7400 pmol/L estrone, with a lower detection limit of 55 pmol/L. An interassay imprecision (CV) of 12-14%, a recovery rate between 80% and 110%, and a dilution linearity are demonstrated. Estrone serum concentrations were measured in healthy men and women and in women with polycystic ovary syndrome. Comparing the assay with a nonextraction RIA, we found an acceptable correlation for samples from 143 subjects of either sex. This enzyme immunoassay with biotin as the primary label and enhanced chemiluminescence signaling detection performs well for determining total estrone in serum and is readily adaptable to assays for other steroid hormones.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.