High-dose hook effect

B.L. Haller, K.A. Fuller, W.S. Brown, J.W. Koenig, B J. Eveland, and M.G. Scott, Two automated prolactin immunoassays evaluated with demonstration of a high-dose hook effect in one. Clin. Chem. 38,... 

The high dose hook effect (HDH) evident as a decrease in signal with very high antigen concentration can be observed in sandwich immunoassays (in particular in single incubation assays). HDH can be prevented by diluting the samples or applying more steps in the assay. 

It is typical for an immunoradiometric assay that a high dose hook effect occurs in the region of very high concentrations. Therefore, sera with concentration above the highest standard S7 have to be diluted with the dilution serum included in the kit. 

The high dose hook effect is a phenomenon observed for one-step sandwich assays at high concentrations of analytes, when different analyte molecules saturate the capture and detection antibodies, respectively [16]. 

A high-dose hook effect that is similar to a prozone phenomenon formerly complicated interpretation of serum ferritin assays specimens with actual ferritin concentration exceeding 1000 pg/L could exhibit count rates that would be obtained at normal serum ferritin concentration. Fortunately, currently available reagents no longer result in the hook effect when serum ferritin concentration is high. 

In order to extend these mathematical models to provide a theoretical basis for the high-dose hook effect, Rodbard et al. (1978) considered two mechanisms (i) heterogeneity of the antibodies immobilized on the solid phase and, (ii) incomplete washing after the first reaction. If two different antibodies are immobilized, AbA and Abe, they both react with the antigen, according to the equations ... 

D Rodbard, Y Feldman, ML Jaffe, LEM Miles. Kinetics of two-site immunoradiometric ( sandwich ) assays. II. Studies on the nature of the high-dose hook effect. Immunochemistry 15 77, 1978. 

FIGURE 3.11 Graphical representation of a high dose Hook effect (HDHE). High levels of analyte can result in the suppression of signal (dotted tine with small closed circles) resulting in a falsely low concentration result. 

Two additional false-negative examples are described below. The first type, the prozone/high-dose hook effect, may occur in lateral-flow assay and one-step sandwich ELISA, which happens when the target protein is present in a sample at a very high concentration. Both capture and labeled antibodies are fully occupied by existing target protein and fail to form the sandwich complex (i.e., labeled-antibody/target... 

---