An improved method for rapid identification of hook effect samples in HBsAg quantitative assay

Abstract

Quantitative hepatitis B surface antigen assay is widely used for diagnosis of hepatitis B virus infection; however, specimens with high levels of the antigen can cause false-negative results (Hook effect), which needs to be resolved. The hook effect samples and non-hook effect samples were detected on the LiCA® 500 instrument using three methods, viz., 1, 2, and 3. Method 1, the currently used procedure, was performed in two steps with a total reaction time of 25 min in a final volume of 250 µL: first incubation was with two reagents for 15 min and then with one other reagent for 10 min. In Method 2, all three reagents were added in one step with a final volume of 250 µL, and the total reaction time was still 25 min. In Method 3, the improved method, all three reagents were added in one step while the final volume was only 130 µL and the total reaction time was only 1 min. Signal values of the non-hook effect samples obtained using Method 2 were significantly lower than those with Method 1, showing competitive inhibition. The hook effect samples tested with Method 2 approximated those obtained using Method 1. Method 3 took 1 min and differentiated hook effect samples successfully, similar to the results with Method 2 which took 25 min. Changing the timing of one reagent addition and incubation time in Method 3 provided a rapid and effective method for the identification of hook effect. The results were more clearly distinguishable due to the phenomenon of competitive inhibition. Method 3 can be considered an improvement on the chemiluminescence platform.