Background: With the advancement of technology, fully automated immunohistochemistry staining systems have been widely adopted in clinical pathological diagnosis due to their standardized operation and high reproducibility. Nevertheless, the specificity and accuracy of staining outcomes are critically dependent on the concentration of the primary antibody. Therefore, the systematic optimization of primary antibody concentration to improve immunohistochemistry staining quality remains a key issue to be addressed. This study aims to optimize the primary antibody concentration for the BOND-III platform to establish standardized protocols and enhance the reliability of immunohistochemistry staining results.

Methods: This study was performed using pathological specimens confirmed positive for a panel of biomarkers. These included the nuclear markers estrogen receptor (ER) and Ki-67, the cytoplasmic markers p16 and cytokeratin 5/6 (CK5/6), and the membranous markers human epidermal growth factor receptor 2 (HER2) and E-cadherin. Immunohistochemical staining was performed using the BOND-III platform to comprehensively assess the impact of a systematic gradient of antibody dilutions on staining quality. The resulting stains were evaluated by comparing the intensity, contrast, and degree of non-specific background across the different dilution levels. Staining scores were statistically compared among dilution groups. Differences in the rate of optimal staining were analyzed using Fisher's exact test.

Results: On the BOND-III platform, the optimal dilution ratio varied significantly for different antibodies. The ER, Ki-67, CK5/6, HER2, and E-cadherin antibodies achieved optimal staining at a 1:2 dilution, characterized by clear positive cell expression, clean background, absence of non-specific staining, and high contrast. Consistently, these five antibodies showed the highest immunohistochemical staining scores at a 1:2 dilution among all tested concentrations, with statistically significant differences compared to the neat group. In contrast, the p16 antibody demonstrated optimal staining at a 1:5 dilution ratio, showing well-defined positive cell localization and clear contrast, which was significantly superior to the neat antibody and all other dilution groups.

Conclusion: Our findings demonstrate that the optimal primary antibody dilution ratio for the BOND-III platform is antibody-dependent. Specifically, antibodies that are often used undiluted on other platforms may require further dilution on the BOND-III, with an optimal range typically falling between 1:2 and 1:5. This optimization not only improves staining quality by enhancing staining specificity and reducing background interference, but also reduces the reagent costs for routine clinical practice.