Hook effect

Another typical problem met in this kind of analysis is known as the hook effect . It is due to an overestimation of the background line to the detriment of the peak tails. As a consequence, the low order Fourier coefficients of the profile are underestimated. In the fitting procedure by pseudo-Voigt functions, this problem occurs if the Gauss content is so high that the second derivative of the Fourier coefficients is negative this is obviously physically impossible because it represents a probability density. 

K.L. Hoffman, G.H. Parsons, L.J. Allerdt, J.M. Brooks, and L.E. Miles, Elimination of hook-effect in two-site immunoradiometric assays by kinetic rate analysis. Clin. Chem. 30, 1499-1501 (1984). 

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,... 

S.A. Fernando and G.S. Wilson, Studies of the hook effect in the one-step sandwich immunoassay. J. 

One common problem of ELISAs affecting accuracy is the hook effect. This is when the signal does not increase with increasing concentration but actually decreases. Another weakness of ELISA is that, compared to other techniques, it has a limited dynamic range. Extrapolation beyond the limits of the range of the standard curve can lead to inaccuracies. 

Thereafter, the different DNA biosensors were used to test PCR samples obtained from F. culmorum. The same DNA biosensor was also challenged with completely non-complementary DNA (Table 29.5). The results exhibited a dose-dependent response up to 7 pg/mL for higher concentrations a hook effect was observed. 

In general, the functional range of a curvilinear regression model is narrower than that of a linear model the assay range is extended by the dilution of samples with concentrations higher than the ULOQ. Dilution linearity also demonstrates the lack of a prozone or hook effect of the assay. A hook (prozone) effect is the phenomenon where the responses of higher concentrations of analyte are lower than expected. 

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 low dose hook effect is a small change in signal in the opposite direction as compared to the normal response-concentration relationship at very low concentrations of analyte [114,115]. In some cases this is attributed to positive allosteric effects for antibody-tracer binding at low concentrations of the analyte. 

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. 

Other technical problems, such as heterophilic antibody (HAMA) interference, may limit the clinical value of Tg measurements. Recommendations regarding approaches to standardization, precision, limits of detection, and hook effects have been suggested. Serum Tg values obtained by different methods are usually not interchangeable, and the same assay should be used to perform serial serum Tg measurements in a patient. When a change in method is made, the laboratory should define performance characteristics of the new method and validate its clinical utility before implementing the method for patient care. 

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 ... 

Although these studies clearly indicate that effective washings are important to prevent the hook effect, the extent of dissociation of the Ab-H-Ab E complex also increases (Section 8.5). Therefore, the elimination of the hook effect may be at the cost of detectability. 

Fig. 13.2. Coating of polystyrene with purified IgG (5 mg/ml) or with complete antiserum in serial dilutions. With the latter a very pronounced optimum of dilution is found, whereas with purified IgG a slight hook effect is observed when tested with the corresponding virus in a sandwich assay. However, the use of purified IgG produces higher detectability than that of complete antiserum for coating. Incubation time required for optimum IgG coating at 37°C was longer than for DNV (Fig. 13.1) (from Tijssen et al., 1982 courtesy of Archives of Virology).

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. 

Dilutional linearity Demonstrate that high-concentration samples can be diluted into range with no hook effect Demonstrate low- and high-concentration samples can be diluted into range with acceptable accuracy and precision Demonstrate low- and high-concentration samples can be diluted into range with acceptable accuracy and precision... 

Most LBAs require some level of sample dilution prior to analysis due to either the assay MRD or high analyte concentrations in the study samples. It is imperative to demonstrate during method development that the analyte, when present in levels above the ULOQ, can be diluted to concentrations within the quantitative range. This may be accomplished by illustrating that the analytical recovery of an ultrahigh matrix spike ( 100 1000 times ULOQ), diluted serially in assay matrix, remains acceptable over a wide concentration range (when corrected for the dilution factor). Dilutional linearity experiments often reveal the presence of a prozone or Hook effect, which is discussed in the next section. 

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. 

The magnitude of the Hook effect in such cases is proportional to the length of incubation until equilibrium is reached. Inadequate wash steps following incubation of the analyte with capture antibody may also lead to leftover free analyte in the assay wells the free analyte can then bind detection antibody, and the complexes removed in... 

FIGURE 3.13 Hook effect revealed by dilutional linearity experiment, (a) The analyte when assayed neat resulted in a low measured concentration. When reassayed at multiple dilutions, the measured concentration for several dilutions was greater than the assay ULOQ. (b) The results, when corrected for the dilution factor, showed dilutional linearity at dilutions greater than 400 fold, indicating the presence of a HDHE. 

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