Estrogen receptor antibody incubation time and extent of immunoreactivity in invasive carcinoma of the breast: The importance of optimizing antibody avidity

We noticed that the percentage and intensity of estrogen receptor (ER) antibody (Ab) AB ER 1D5 immunohistochemical (IHC) staining was altered by Ab incubation time and the type of chromogen detection system in invasive breast carcinomas. We studied the impact of these 2 factors on Ab ER 1D5 immunoreactivity. Serial sections from 22 strongly ER-positive invasive breast carcinomas were immunohistochemically stained with Ab clone ER 1D5 using 3 IHC protocols. One IHC protocol used a 12-hour Ab incubation and a supersensitive, labeled streptavidin-biotin chromogen detection system (12 h-Standard), the second IHC protocol used a 2-hour Ab incubation and a supersensitive, labeled streptavidin-biotin chromogen detection system (2h-SS), and the third protocol used a 2-hour Ab incubation and a polymer-based detection system (2h-Env). Twenty identical fields on each slide stained with each IHC protocol were evaluated and staining was quantified using image analysis. The mean staining percentages using the 12h-Standard, 2h-SS, and 2h-Env IHC staining protocols were 89%, 72%, and 47%, respectively (P<0.001). Three of the 22 cases (14%) were ER negative ( < 10% stained area) with the 2h-Env IHC protocol. Stain intensity was significantly stronger with the 12h-Standard Ab incubation IHC protocol than either 2-hour Ab incubation protocol (P < 0.001). Twelve cases stained with 2-hour Ab incubation IHC protocols had weak visually seen staining: 7 were Allred total score 2 (ER negative) and 5 were Allred total score 3. Ab ER 1D5 avidity is directly related to factors that impact electrostatic forces, one of which is Ab incubation time. Standard automated stainer Ab incubation times of less than I hour may be of insufficient duration and result in artificially low levels of ER immunoreactivity. The chromogen detection system in association with the ER 1D5 Ab also alters levels of immuno reactivity. Optimization of IHC staining protocols should include evaluating the Ab incubation time and chromogen detection system. These factors can substantially alter the extent and intensity of ER IHC staining.