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Step 1: Antigen Retrieval: [ Ссылка ]
Step 2: Primary Antibody Diluent: [ Ссылка ]
Step 3: Detection Reagents (this video)
Step 4: Chromogen
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Step 3 of our Better IHC series focuses on the reagents that catalyze chromogen deposition to visualize proteins, and compares biotin-streptavidin detection systems to polymer-based detection.
Transcript:
Step three. Detection Reagents.
Detection reagents carry enzymes to the site of the specific epitope by binding to the primary antibody, either directly or indirectly, through a secondary antibody intermediate. When a chromogenic substate for HRP is introduced, a precipitate forms that deposits at the site of the primary antibody antigen binding event, making it visible upon microscopic examination.
Historically, this interaction is facilitated by biotin and streptavidin. The secondary antibody is conjugated to biotin, which can bind streptavidin, and the streptavidin molecule itself is conjugated to HRP.
This method has several limitations.
First, the streptavidin-HRP complex can bind to endogenous biotin and produce significant background signal. Endogenous biotin can be exacerbated if Heat Induced Epitope Retrieval is used, as buried biotin is unmasked alongside other epitopes.
Second, the intensity of the chromogenic signal is determined by the amount of enzyme present, but the number of HRP molecules bound to the site of the primary antibody-antigen interaction is restricted to the number of streptavidin molecules bound to the complex.
Polymer-based systems are gaining in popularity, because they avoid the limitations of the biotin-based system. In this method, a polymer, like dextran, is conjugated to a secondary antibody and the HRP enzymes simultaneously. The polymer backbone eliminates the need for building biotin-streptavidin-HRP complexes at the primary antibody antigen site, eliminating the potential for biotin-based background noise. Plus, the number of HRP molecules that be bound is higher relative to the streptavidin system, so fewer primary antibody antigen binding sites produce proportionately more signal, increasing the sensitivity of the assay.
When we used a biotin-based detection system to evaluate the PLK1 Rabbit mAb, we found it didn't provide a strong enough signal, even with the other changes in companion reagents we had made. As a result, we switched to the more sensitive polymer-based detection method.
Despite an appreciable increase, it was not sufficient to meet our standards. But, we had one more trick up our sleeve.
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