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Objectives: The therapeutic monoclonal antibody (t-mAb) daratumumab, used to treat multiple myeloma (MM) patients, interferes with routine, electrophoretic based M-protein diagnostics. Electrophoretic response assessment becomes increasingly difficult when multiple t-mAbs are combined for use in a single patient. This is the first study to address the analytical challenges of M-protein monitoring when multiple t-mAbs are combined., Methods: In this proof-of-principle study we evaluate two different methods to monitor M-protein responses in three MM patients, who receive both daratumumab and nivolumab. The double hydrashift assay aims to resolve t-mAb interference on immunofixation. The MS-MRD (mass spectrometry minimal residual disease) assay measures clonotypic peptides to quantitate both M-protein and t-mAb concentrations., Results: After exposure to daratumumab and nivolumab, both t-mAbs become visible on immunofixation electrophoresis (IFE) as two IgG-kappa bands that migrate close to each other at the cathodal end of the γ-region. In case the M-protein co-migrates with these t-mAbs, the observed interference was completely abolished with the double IFE hydrashift assay. In all three patients the MS-MRD assay was also able to distinguish the M-protein from the t-mAbs. Additional advantage of the MS-MRD assay is that this multiplex assay is more sensitive and allows quantitative M-protein-, daratumumab- and nivolumab-monitoring., Conclusions: Daratumumab and nivolumab interfere with electrophoretic M-protein diagnostics. However, the M-protein can be distinguished from both t-mAbs by use of a double hydrashift assay. The MS-MRD assay provides an alternative method that allows sensitive and simultaneous quantitative monitoring of both the M-protein and t-mAbs., (© 2021 Somayya Noori et al., published by De Gruyter, Berlin/Boston.)

We have previously shown that different individuals exposed to the same antigen produce antibodies with identical mutations in their complementarity determining regions (CDR), suggesting that CDR tryptic peptides can serve as biomarkers for disease diagnosis and prognosis. Complete Fabs derived from disease specific antibodies have even higher potential; they could potentially be used for disease treatment and are required to identify the antigens toward which the antibodies are directed. However, complete Fab sequence characterization via LC-MS analysis of tryptic peptides (i.e. bottom-up) has proven to be impractical for mixtures of antibodies. To tackle this challenge, we have developed an integrated bottom-up and top-down MS approach, employing 2D chromatography coupled with Fourier transform mass spectrometry (FTMS), and applied this approach for full characterization of the variable parts of two pharmaceutical monoclonal antibodies with sensitivity comparable to the bottom-up standard. These efforts represent an essential step toward the identification of disease specific antibodies in patient samples with potentially significant clinical impact., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Paraneoplastic neurological syndromes (PNS) are devastating neurological disorders secondary to cancer, associated with onconeural autoantibodies. Such antibodies are directed against neuronal antigens aberrantly expressed by the tumor. The detection of onconeural antibodies in a patient is extremely important in diagnosing a neurological syndrome as paraneoplastic (70% is not yet known to have cancer) and in directing the search for the underlying neoplasm. At present six onconeural antibodies are considered 'well characterized' and recognize the antigens HuD, CDR62 (Yo), amphiphysin, CRMP-5 (CV2), NOVA-1 (Ri), and Ma2. The gold standard of detection is the characteristic immunohistochemical staining pattern on brain tissue sections combined with confirmation by immunoblotting using recombinant purified proteins. Since all six onconeural antibodies are usually analyzed simultaneously and objective cut-off values for these analyses are warranted, we developed a multiplex assay based on Luminex technology. Reaction of serial dilutions of six onconeural standard sera with microsphere-bound antigens showed lower limits of detection than with Western blotting. Using the six standard sera at a dilution of 1:200, the average within-run coefficient of variation (CV) was 4% (range 1.9-7.3%). The average between-run within-day CV was 5.1% (range 2.9-6.7%) while the average between-day CV was 8.1% (range 2.8-11.6%). The shelf-life of the antigen coupled microspheres was at least two months. The sensitivity of the multiplex assay ranged from 83% (Ri) to 100% (Yo, amphiphysin, CV2) and the specificity from 96% (CV2) to 100% (Ri). In conclusion, Luminex-based multiplex serology is highly reproducible with high sensitivity and specificity for the detection of onconeural antibodies. Conventional immunoblotting for diagnosis of onconeural antibodies in the setting of a routine laboratory may be replaced by this novel, robust technology., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Paraneoplastic neurological syndromes (PNS) are severe immune mediated effects of cancer. The presence of IgG autoantibodies against onconeural antigens in serum is a hallmark of the disease. Multiple paraneoplastic antibodies have been described, including antibodies against HuD, Yo, amphiphysin and CV2. In this study, we test the hypothesis that primary amino-acid structures of the antigen binding part of antibodies from various individuals share common sequences that are specific for each auto-antigen. We selected 60 patients with PNS, associated with antibodies against HuD, Yo, Amp or CV2. Affinity purified IgG was separated using SDS-PAGE and IgG heavy chains were excised, trypsinized and subjected to tandem mass spectrometry. We selected masses that uniquely identified a PNS autoantibody group, and used MS/MS fragmentation spectra to obtain information on peptide sequences. Out of 19,173 unique masses, 28 immunoglobulin-derived peptides were found exclusively in samples from a single autoantibody defined PNS group. Our results confirm that specific peptide structures exist in the antigen binding site of IgG that are shared between individuals harboring autoantibodies against the same onconeural antigen. Thus, the immune response in these patients followed converging paths during the rearrangement, selection and maturation of immunoglobulin sequences. The identified peptides can be applied in the diagnosis of PNS, but these data also indicate that a similar approach in a variety of other diseases involving an immune response would have an appealing outlook., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We present a new procedure that allows for the controlled patterning of functional proteins on the sidewalls of three-dimensional microfabricated chambers. We build microchambers with walls containing gold areas of a defined submicrometer size, to which proteins can be bound specifically, via thiol chemistry. We tested our system by observing the gliding of microtubules along motor-coated microchamber sidewalls and find that introducing an intermediate multilayer of proteins greatly enhances motor protein activity. This combination of spatial confinement and three-dimensional patterning of protein activity opens up new possibilities for the in vitro study of cellular processes, as well as for the development of guided transport mechanisms in nanodevices.

The tubular morphology of intracellular membranous compartments is actively maintained through interactions with motor proteins and the cytoskeleton. Moving along cytoskeletal elements, motor proteins exert forces on the membranes to which they are attached, resulting in the formation of membrane tubes and tubular networks. To study the formation of membrane tubes by motor proteins, we developed an in vitro assay consisting of purified kinesin proteins directly linked to the lipids of giant unilamellar vesicles. When the vesicles are brought into contact with a network of immobilized microtubules, membrane tubes and tubular networks are formed. Through systematic variation of the kinesin concentration and membrane composition we study the mechanism involved. We show that a threshold concentration of motor proteins is needed and that a low membrane tension facilitates tube formation. Forces involved in tube formation were measured directly with optical tweezers and are shown to depend only on the tension and bending rigidity of the membrane. The forces were found to be higher than can be generated by individual motor proteins, indicating that multiple motors were working together to pull tubes. We propose a simple mechanism by which individual motor proteins can dynamically associate into clusters that provide the force needed for the formation of tubes, explaining why, in contrast to earlier findings [Roux, A., Cappello, G., Cartaud, J., Prost, J., Goud, B. & Bassereau, P. (2002) Proc. Natl. Acad. Sci. USA 99, 5394-5399], motor proteins do not need to be physically linked to each other to be able to pull tubes.