ELIMINATION OF NON‐SPECIFIC SIGNALS

According to the results of the experiments described above, the positive signal (non-specific signal) level for negative control experiments was repeatedly persistent. It showed no significant difference in negative control signal between different host species therefore non-specific signal is not due to discrepancy of cross reactivity of different PLA probes.

Also decreasing the primary concentration up to 5000 fold did not help to decrease the non-specific signal.

So at this point, it was decided to optimize the PLA protocol further to be able to decrease non-specific signals and/or decipher the underlying mechanisms. There were two main aspects to be yet confirmed: either inefficient washing step or insufficient blocking step causing the excessive non-specific interactions. Therefore, several attempts to remove these nonspecific signals were done by changing the volume of washing solution and applying additional blocking incubation step.

INCREASING VOLUME OF WASHING SOLUTION

Firstly, how volume of washing solutions would affect the non-specific signal was investigated. It was suspected that the washing steps after PLA probe incubation was not efficient to remove excess PLA probe due to the lower volume of solutions used in hybridization chambers that can take 40 μL. These chambers were used for practicality and in previous experiments, it had not been removed between any steps so for each washing step, 40μl of solution had been used. However, Olink protocol suggests using 70ml of solution for each washing step. It had been kept used before, because there was no signal observed on the surface on the glass slide, outside of the cell cytoplasm suggesting that lower volume of washing solution does not cause non-specific interaction. But it might be critical for non-specific interaction on cell surface so the volume of washing was increased as suggested and compared with using 40μl of washing volume. Results and conditions for Olink protocol and the one used in our experiments are depicted in tables below. Note that also time of washing step had been increased in contrast to recommended protocol which might have an effect in kinetics of dissolution of antibodies.

.TABLE 4-2-10 COMPARISON FOR NON-SPECIFIC PLA SIGNAL WITH HIGH AND LOW VOLUME OF WASHING SOLUTION, SIGNALS WERE QUANTIFIED WITH CELL PROFILER WITH SAME PARAMETERS WITHIN THE SAME EXPERIMENTS, N IS THE NUMBER OF CELLS USED FOR COUNTING, P IS THE PASSAGE NUMBER.

Cell  Exp.  Her2+Her3+  Her2+Her3‐  Her2‐Her3+  Her2‐Her3‐ 

SKBR3 (p14)  S9 (40µl)  78,7 (N=76)  29,5 (N=73)  ‐  ‐ 

SKBR3 (p17)  S12 (70ml)  53,6 (N=52)  36,1 (N=54)  1,3 (N=54)  0,5 (N=64)  Unfortunately these results did not show any improvement when using high volume of washing solution (Table 4-2-10). It was obtained comparable signal quantities for each

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case, 29 for low volume and 36 for high volume. In any case, it was decided to keep using high volume for washing step after the PLA probe incubation for next experiments.

TABLE 4-2-11 CONDITIONS USED IN EXPERIMENTS AND IN RECOMMENDED OLINK PROTOCOL EXP.  Blocking 

ADDING EXTRA BLOCKING STEP BEFORE PLA PROBE INCUBATION

As a second option, it was thought that additional blocking step before incubating PLA probes could help to decrease the non-specific interactions of secondary antibodies. So a blocking step for 30 minutes was added to the protocol with the same blocking solution used for 1° ab provided by kit.

TABLE 4- 2-12 COMPARISON FOR NON-SPECIFIC PLA SIGNAL WITH ADDITIONAL BLOCKING STEP (S15) OR NOT (S14), SIGNALS WERE QUANTIFIED WITH CELL PROFILER WITH SAME PARAMETERS FOR BOTH CONDITION; N IS THE NUMBER OF CELLS USED FOR COUNTING

Cell  Exp.  Her2+Her3+  Her2+Her3‐  Her2‐Her3+  Her2‐Her3‐ 

SKBR3 (p2)  S14  47,8 (N=53)  5,9 (N=59)  0,1 (N=80)  0,08(N=78)  SKBR3 

(p2)Blocking step  S15  64,2 (N=60)  9,7 (N=73)  0,2(N= 84)  0,1 (N=56)  Standard 

Deviation    11,67  2,69  0,07  0,02 

This experiment (S14 and S15) was performed as parallel with the same passage of cells and repeated only once. However, it was concluded that adding blocking step before PLA probe incubation did not improve to remove non-specific signal (Table 4-2-12). On the contrary, it seems that it had slightly increased the number of non-specific signal and also positive signal. So it was decided to continue to use the previous protocol as S12.

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As a summary of section 4.2.3, we faced serious problems of non-specific generation of PLA “spots”, making the definition of a reliable threshold between “positive” and

“negative” cell line problematic. Thorough attempts to adapt the protocol conditions to reduce this non-specific signal, using the strategy proposed by the kit provider, did not provide a solution to this problem. The overall protocol involve enough steps, so that room for testing further modifications is virtually endless, and we cannot ascertain that solutions could not be found by further work in this direction. It seemed to us, however, that this strategy involved serious risk of accumulating vast amounts of work with no guarantee of success, since the molecular origin of the problem remains unknown. The indirect PLA method involves using secondary antibodies with oligonucleotide conjugates directed against the host species of a primary antibody bind to the antigen of interest. This strategy has its advantages, in particular in terms of production, and not requiring conjugating primary antibodies altering its affinity thus a large number of commercial available primary antibodies can be adapted to a large number of antigens. However, one has essentially no control on the detailed epitopes of secondary antibodies from different species, and we concluded above that cross-reactivity between these primary and secondary antibodies could very plausibly be the main cause of our problems. We thus decided to use direct PLA strategy, based on a less versatile but more specific approach, directly conjugating PLA probes on primary antibodies to HER2 and HER3, respectively using commercially available Probe Maker kit.

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4.2.5 DIRECT PLA WITH PRIMARY CONJUGATES FOR HER2:HER3