Monitoring protein-tissue interactions in real time

Saturday, 02 January, 2010

Ridgeview Instruments AB’s LigandTracer Grey facilitates kinetic measurements on living cells by enabling researchers to follow protein-cell interactions in real time.

The analysis of how proteins interact with tissue is currently performed using immunohistochemistry (IHC), a semiquantitative binding assay which relies on staining of antibodies specific for selected receptors. This application note shows how LigandTracer can be used for real-time detection of how antibodies (Abs) interact with tissue, both as labelled primary Abs and by use of secondary Abs.

Experimental

In a first example, a paraffin-embedded SKOV-3 xenograft tumour from a mouse was placed on a pretreated glass petri dish, was de-paraffinised and heat-treated for epitope retrieval.

The glass dish was placed in LigandTracer Grey and an anti-HER2 Ab (DAKO, Glostrup, Denmark) labelled with ¹²⁵I was added (1 nM). After 20 hours of incubation, unlabelled anti-HER2 Ab was added at a total concentration of 10 nM.

In a second example, paraffin-embedded tissue from three different sources (breast from a breast cancer patient, tonsil from a healthy subject and placenta from a healthy subject) was (i) placed on a pretreated glass dish, (ii) de-paraffinised, (iii) heat-treated for epitope retrieval and (iv) incubated with a primary anti-HER2 AB at 1 nM (DAKO) for one hour. After thorough wash, the glass dish was positioned in a LigandTracer Green prototype at room temperature and FITC-labelled secondary Ab directed against the primary Ab was added to a final concentration of 40 nM. A consecutive tissue section was stained according to standard IHC protocols for comparison.

Results

Figure 1 shows the primary interaction of radiolabelled anti-HER2 Ab (DAKO) to SKOV-3 xenograft. There is a clear binding phase during incubation and it takes more than 20 hours to approach equilibrium for this interaction at 1 nM of the Ab.

Figure 1: ¹²⁵I - Anti-HER2 antibody binding to SKOV-3 xenograft followed by competition with unlabelled antibody.

Upon addition of unlabelled Ab, the signal level decreases as a result of competition for the binding sites. This decrease is a strong indication of a specific interaction.

Figure 2 shows how the secondary Ab binds to the tissue sections in the glass dishes. When incubation media is replaced with buffer the beginning of wash-out is seen. The real-time interaction pattern after incubation with secondary Ab is similar to the IHC staining results.

Figure 2: LigandTracer interaction analysis of how the secondary Ab bind to tissue pre-incubated with primary Ab.