Dilution Protocols for Detection of Hook Effects/Prozone Phenomenon

The prozone or (high-dose) hook effect, documented to cause false-negative assay results >50 years ago (1), still remains a problem in one-step immunometric assays (2)(3)(4)(5)(6)(7)(8)(9), immunoturbidimetric assays (10), and immunonephelometric assays (11) for immunoglobulins. To detect the prozone effect, samples are often tested undiluted and after dilution (9). If the result on dilution is higher than for the undiluted sample, then the undiluted sample most likely exhibited the prozone effect. Unfortunately, this approach increases labor and reagent costs for assays that may only rarely encounter extremely high analyte concentrations. An alternative approach involves pooling patient samples and measuring the pool and a 10-fold dilution of the pool (12). If one or more of the samples in the pool is falsely low because of the prozone effect, then the results from the undiluted and diluted pools (after correcting for the 10-fold dilution) will differ significantly (12). Other approaches to eliminate the prozone effect include using two-step immunoassays that have a wash step between the addition of …