Comparison of Nanobodies to Classical Affinity Probes

The limited possibility to use antibodies as a tool for protein quantification is only one difference to nanobodies. As pointed out in section 1.5 and supported by my findings in section 3.5, antibodies are impaired by their size from penetration deep into complex biological samples as demonstrated in section 3.5.3. Consequently, they might not detect all available target epitopes yielding an inhomogeneous staining and thus not necessarily reflecting the actual protein distribution in the sample [72] as shown in section 3.5.5.

Typically, biological samples are chemically fixed prior to immunostainings using aldehydes such as PFA or glutaraldehyde. However, the fixation is often not completed as the aldehydes may take up to several days to achieve complete immobilization of the molecules in the sample [189,190].

Moreover, the crosslinking induced by the aldehydes can be reversed if the fixation was performed only shortly and the sample is left in an aqueous solution. Therefore divalent probes such as antibodies might cluster together insufficiently immobilized antigens upon binding. This effect is even enhanced by the use of secondary antibodies for detection as the sample is commonly not fixed again after application of the primary antibody. Divalent polyclonal antibodies such as secondaries can bind multiple primary antibodies, which might thus increase the clustering effects.

Figure 28 shows the difference in phenotype when using primary and secondary antibodies or directly conjugated nanobodies to detect endogenous proteins. My data suggest that using antibodies not only results in the tendency to observe more bright spots, but might result in omitting antigens in between these spots. As a consequence, the IF image does not necessarily represent the biological distribution of the antigen. In contrast, nanobodies used in IF experiments generally showed a more smooth antigen distribution as shown in section 3.5.

These effects can merely be seen in confocal microscopy, but they become a major problem in super-resolution microscopy [91,105]. At a diffraction-limited super-resolution, the size of antibody clusters is expected to be well below the maximum attainable resolution of 200 nm resulting in a similar staining pattern compared to nanobodies [91,105]. If using current super-resolution techniques, the technically possible resolution is compromised by the size of the antibody as illustrated in Figure 32.

Modern microscopes for STED and STORM can achieve resolutions in the low nanometer range, whereas the size of a single antibody molecule already ranges around 15 nm [12,27,111].

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In addition to the resulting fluorophore delocalization, the molecular clustering and inhomogeneous fluorescent labeling of the secondary antibody are prone to distort the phenotype of antigen organization (see section 1.5). In contrast, a monovalent small affinity probe is able to efficiently decorate the target antigen resulting in a better resolved staining resembling the antigen distribution in super-resolution (represented by blurred red circles in Figure 32A). Although Figure 32 is a simplified 2D-projection, it illustrates the problem for one exemplary protein. In a complex biological sample, not only special dimensions of the depicted proteins but also the densely packed molecular environment needs to be considered as depicted in section 1.6.

In such an environment, the epitopes of the target antigen might be masked by other proteins and thus are not accessible for antibodies. The flexible CDR3 loop of nanobodies (see section 1.4.4) in combination with the small size of the probe can be beneficial in such a case.

However, the missing of epitopes might also happen to nanobodies if the antigen is engaged in a multimeric complex as it might be the case for S25-Nb10 where the epitope is located within the SNARE domain (see Figure 23). This would also impair nanobodies from accessing the epitope in assembled SNARE complexes, which could be an explanation for the low degree of colocalization observed in Figure 30.

Despite the increase in resolution achieved by using directly conjugated affinity probes, this detection system is to some extend rather inflexible. If an alternative fluorescent label is desired, the molecule needs to be labeled de novo with the dye. In contrast, the label in an antibody staining can easily be exchanged by using an alternative secondary antibody providing a flexible platform for fluorescent labeling. However the conjugation of nanobodies to functionalized dyes can be performed with reasonable effort following standard protocols. The conjugation to recombinant tags or enzymes is even facilitated as they can directly be fused to the nanobody on DNA level. Along these lines, nanobodies might be useful tools to replace secondary antibodies to compensate for potential artifacts introduced while performing conventional antibody stainings.

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Figure 32: Schematic illustration of antibody and nanobody binding to a target antigen. A: Exemplary representation of fluorescence signals detected by microscopy. With super-resolution techniques, individual fluorophores (blurred red circles) can be localized indicating the arrangement of the target antigen. Notably, when using confocal microscopy, the diffraction limit would cause the fluorescent signals to overlap impeding conclusions on molecular organization (represented by the dashed grey line). B: A potential molecular arrangement of affinity probes resulting in a fluorescence pattern observed in A. Although the detected fluorescence pattern resulting from antibody or nanobody staining is different, the molecular organization of the antigen (SNAP-25) is still identical. The fluorescence signal resulting from the nanobody resembles the molecular organization of the antigen with higher accuracy thus increasing the resolution.

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The scalability of nanobody production further broadens the possible applications as experiments are typically not limited by the amount of affinity probe available. Hybridoma cell lines for the production of monoclonal antibodies are cost-intensive and might eventually die or stop the secretion of functional antibodies [107]. Although polyclonal antibodies are more easy to produce then monoclonal antibodies, they often show high batch variability in binding to their antigen resulting in several researches recommending to substitute them [107,191].

Those antibody characteristics often associated with artifacts in IF experiments are not observed when using nanobodies as affinity probes. Their small size and monovalent nature facilitate the efficient decoration of cellular epitopes thus providing a higher level of detail than conventional affinity probes. If introducing the fluorescent label in a quantitative manner, the microscopy image can even be assumed to directly reflect the actual distribution of the antigen. However, the labeling of nanobodies requires careful optimization as mentioned above. If only one dye molecule is attached per nanobody, the absolute fluorescence signal is essentially lower than observed with secondary antibodies, which are known to amplify the signal.

On the other hand, the high detection rate of the nanobody somewhat compensates for this effect as the number of fluorescent molecules is higher compared to conventional antibodies. If the antigen is present in plethora, using nanobodies might be even more reliable as the higher fluorescence of antibodies is counteracted by their poor penetration capability (see section 3.5.4). During the studies, I primarily used one antibody clone for each SNAP-25 and syntaxin 1A for direct comparison with my nanobodies. Although it might be interesting to analyze also the performance of other antibodies, I chose the clones 71.1 and 78.2 as two very established antibodies in the field [128,165,192]. As the aforementioned observations are due to general properties of antibodies, also alternative clones are likely to yield the same result.

I observed significant differences in the fluorescence signal revealed by antibodies or nanobodies in both cell lines and primary neuron cultures. The accumulation of overexpressed syntaxin 1A in the ER and Golgi if expressed without cofactors has been reported before [193]. This explains the phenotype in the EGFP-channel showing high concentration of syntaxin 1A in the perinuclear region as depicted in section 3.5.2. Similarly, SNAP-25 was found in higher concentrations at the periphery of the nucleus.

After expression, SNAP-25 is palmitoylated to be anchored into cellular membranes. Enzymes of the DHHC family that mediate the palmitoylation of SNAP-25 were reported to predominantly be located at the Golgi apparatus, which resembles the structure and location of high SNAP-25-EGFP signal in COS-7 cells [194]. The high copy numbers of the overexpressed protein presumably saturate the capacity of the cellular palmitoylation machinery and cause the protein to accumulate. In both cases,

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the antigen is localized in a protein-dense region, where the nanobodies represent the EGFP signal significantly better compared to conventional antibodies.

A similar phenotype could also be observed when staining PC-12 cells with either antibodies or nanobodies, shown in section 3.5.4. As those cells originate from the neural crest, they endogenously express both SNAP-25 and syntaxin 1A, albeit in lower amount than in an overexpressing system [192]. This suggests that at the level of endogenous protein expression, the conventional antibodies used in this experiment cannot reliably detect the entirety of present antigens and their organization. This is a crucial point particular for the detection of the SNARE proteins SNAP-25 and syntaxin 1A in neuronal synapses as they play a major role in synaptic vesicle fusion and neurotransmitter release [126]. It is therefore of major interest to analyze the organization of those proteins in the highest level of detail possible, which can only partially be done with conventional antibodies.

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