Prozone effect

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. 

Steric hindrance due to high antigen level and possible prozone effect. Re-optimize concentration of the primary antibody and ancillary reagents. Antibody concentration of the primary antibody may be too high. 57-60... 

It is very important in PAP techniques for the secondary antibody to be applied in excess. This way, one arm of the divalent Fab portion of the immunoglobulin molecule can bind the primary antibody, and the other arm is free to bind the PAP complex. If not in excess, both arms of the molecule may bind the primary antibody, creating a prozone-type of effect. Without a free secondary antibody Fab site to capture the PAP complex, the assay will not work. 

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. 

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. 

Dilutional Linearity. As the range of an immunoassay is usually limited, it may be necessary to dilute concentrated samples. Therefore, it has to be demonstrated that the analyte can still be reliably quantified upon dilution of high concentration samples so that they fall within the validated range of the assay. Moreover, a so-called prozone or hook effect can be identified. A hook effect is present when high concentration samples above the ULQQ display a lower response than ULQQ samples because of signal suppression caused by the high concentration of the analyte [30]. Dilutional linearity should not be confused with parallelism (see below). 

Numerous variables can lead to a negative result from passive immunotherapy. Too little antibody can result in a lack of protective efficiency. Too much antibody can produce a prozone-like effect, whereby more antibody provides less protection than less antibody. The antibodies used in therapy can also be affected by affin-ity/avidity, specificity, isotype, idiotype, preparation, and route of administration. The genetic background and immune competency of the host can also affect treatment as can route of pathogen infection, genetic background, and inoculum size of the pathogen [114]. A dramatic example of the limitations of passive antibody transfer experiments is provided by the observation that transfer of either too little or too much antibody can result in no protection [114]. 

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... 

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