Understanding the Hook Effect in a One-Step Sandwich ELISA


The Enzyme-Linked Immunosorbent Assay (ELISA) is a popular method used to detect and quantify soluble analytes such as proteins, hormones, and antibodies (1, 2). Despite its widespread application and high sensitivity, one notable limitation is the Hook Effect in a one-step assay protocol, a phenomenon that can lead to significantly inaccurate results in assays, especially at very high concentrations of the target analyte.

What is the Hook Effect?

The Hook Effect, also known as the prozone or High Dose Hook Effect, occurs when extremely high concentrations of the target analyte cause a paradoxical decrease in signal detection. Instead of a linear or sigmoid detection curve, the signal initially increases with analyte concentration, but then decreases, resulting in a falsely low signal. This effect can result in a false negative or underestimation of analyte concentration (3).

 To comprehend the mechanism of the Hook Effect, it's important to understand the basic principles of the ELISA procedure.  In a one-step sandwich ELISA, the capture antibody is immobilized on a solid surface and both the analyte and the enzyme-linked detection antibody are added in the same step. The expectation is that the analytes are sandwiched between the capture and detection antibodies, resulting in a color change upon the addition of a substrate which interacts with the enzyme-linked detection antibody.  However, at very high analyte concentrations, excess analyte saturates the capture antibodies, leaving free analyte in the assay solution. The free analyte in solution competes with the captured analyte for binding to the detection antibodies. These conditions reduce the effective binding of the detection antibodies, resulting in a lower signal despite the high analyte concentration.


Using Chondrex, Inc.'s Type II Collagen Detection Kit (Cat # 6018) with its one-step ELISA assay protocol, the Hook Effect was demonstrated in Figure 1 and Table 1. The linear assay range (reportable range) of the ELISA is 3.1 - 200 ng/ml, the error range is 300-3000 ng/ml, and the Hook Effect occurs at concentrations greater than 3000 ng/ml.

Figure 1. The Hook Effect in the Type II Collagen Detection ELISA Kit using the one-step assay protocol.

Table 1. The Hook Effect depends on type II collagen concentration in the Type II Collagen Detection ELISA Kit using the one-step assay protocol.

Two Strategies Can Mitigate the Hook Effect:

1. Sample Dilution: preparing a wide range of sample dilutions to determine where it lies within the assay's linear range.

2. Two-step assay protocol: separating the capture and detection reaction steps can eliminate the competitive reactions. Chondrex, Inc. provides a two-step assay protocol for Cat # 6018 in Figure 2.

Figure 2. Assay Outline


The Hook Effect is a critical consideration in ELISA assays, especially in research settings where accurate quantification is critical. By understanding the mechanism, recognizing the signs, and implementing mitigation strategies, scientists can minimize its impact and ensure reliable results. Careful experimental design and validation are key to overcoming this challenge and realizing the full potential of ELISAs in detecting and quantifying target analytes.

Understanding the Hook Effect and its implications allows for better interpretation of ELISA results, ultimately leading to more accurate diagnoses and research findings. As with any analytical technique, awareness of potential pitfalls and proactive measures can significantly improve the robustness and reliability of the data obtained.

For more information, please refer to the Type II Collagen Detection Kit (Cat # 6018) .


  1. D. Wild, The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques. Elsevier (2005).
  2. J. Gosling, A decade of development in immunoassay methodology. Clinical Chemistry, 36(8), 1408-1427 (1990).
  3. A.  Butch, Dilution protocols for detection of hook effects/prozone phenomenon. Clin. Chem. 46, 1719–1721 (2000).


Join Our Mailing List