A novel polymeric labeling strategy allows preparation of compact enzyme antibody conjugates that offer increased detection sensitivity and efficiency, while simplifying staining procedures. Such a conjugated reagent, designated the PowerVision system, was evaluated to assess its detection efficiency and sensitivity, based on a comparative study with three currently available multistep detection systems: ChemMate, LSAB2, and Super Sensitive kits. All immunohistochemical staining was performed on routinely formalin- fixed, paraffin-embedded tissue sections under identical conditions, including use of the microwave antigen-retrieval (AR) technique. The experimental design incorporated three sets of experimental conditions to facilitate comparison of the different methods. Experimental set 1, comparing the immunostaining results obtained using a panel of 15 antibodies revealed that the sensitivity of the PowerVision detection system was significantly greater than the other three methods, as evidenced by the greater intensity of immunostaining achieved for all 15 antibodies. Experimental set 2, an efficiency test, used further serial dilutions of a commercial prediluted, ready-to-use monoclonal antibody (Mab) to proliferation cell nuclear antigen (PCNA). Dilutions ranged from 1:10 to 1:640. The detection efficiency of the PowerVision reagent was superior to the other detection systems, as exemplified by the observation that at a dilution of 1:320, a moderate staining intensity could be achieved by using the PowerVision system, while other methods showed faintly positive or negative results. Experimental set 3 demonstrated that the immunoreactivity of long-term-stored archival paraffin sections for Mab to p27Kip1 could be restored to a similar level to that obtained using fresh-cut paraffin sections, when combining an optimized AR protocol [using ethylenediaminetetraacetic acid-sodium hydroxide (EDTA-NaOH) solution of pH 8.0] with the PowerVision system, whereas other reagents gave unsatisfactory results. The controlled polylabeling PowerVision detection system has the advantages of a simpler, more rapid procedure, significant amplification of staining intensity, lower costs resulting from further dilution of primary antibodies, faster turnaround, and higher amplification power. It is also a biotin-free reagent avoiding the problems of endogenous biotin reaction.
- Antigen retrieval
- Polymeric conjugates