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Comprehensive molecular characterization of a cisplatin-specific monoclonal antibody Lena Ruhe, Stefanie Ickert, Ulrike Hochkirch, Johanna Hofmann, Sebastian Beck, Jürgen Thomale, and Michael W. Linscheid Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00575 • Publication Date (Web): 11 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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Molecular Pharmaceutics

Comprehensive molecular characterization of a cisplatinspecific monoclonal antibody Lena Ruhe1, §, #, Stefanie Ickert1, 2, #, Ulrike Hochkirch1, Johanna Hofmann3, Sebastian Beck1, Jürgen Thomale4 and Michael W. Linscheid1,* 1

Humboldt-Universitaet zu Berlin, Department of Chemistry, Brook-Taylor-Str. 2, 12489 Berlin

2

Federal Institute for Materials Research and Testing, Richard-Willstaetter-Str. 11, 12489 Berlin

3

Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195 Berlin

4

Universitaetsklinikum Essen, Department of Cell Biology, Hufelandstr. 55, 45122 Essen

§

Present address: Labor Berlin-Charité Vivantes GmbH, Sylter Str. 2, 13353 Berlin

# both authors contributed equally * Correspondence should be addressed to [email protected], Phone: +49 30 2093 7588; Fax: +49 30 2093 6985

Abstract Despite their immense and rapidly increasing importance as analytical tools or therapeutic drugs, the detailed structural features of particular monoclonal antibodies are widely unknown. Here, an antibody already in use for diagnostic purposes and for molecular dosimetry studies in cancer therapy with very high affinity and specificity for cisplatin-induced DNA modifications was studied extensively. The molecular structure and modifications as well as the antigen specificity were investigated mainly by mass spectrometry. Using nano electrospray ionization-mass spectrometry, it was possible to characterize the antibody in its native state. Tandem-MS experiments revealed not only specific fragments, but also gave information on the molecular structure. The detailed primary structure was further elucidated by proteolytic treatment with a selection of enzymes and high resolution tandem-MS. The data were validated by comparison with known antibody sequences. Then, the complex glycan structures bound to 1 ACS Paragon Plus Environment


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the antibody were characterized in all detail. The Fc-bound oligosaccharides were released enzymatically and studied by matrix-assisted laser desorption/ionization - mass spectrometry. Overall 16 different major glycan structures were identified. The binding specificity of the antibody was investigated by applying synthetic single and double stranded DNA oligomers harboring distinct Pt adducts. The antibody-antigen-complexes were analyzed by mass spectrometry under native conditions. The stability of the complex with double stranded DNA was also investigated.

Keywords: Antibody – Native MS – Cisplatin - PTM

Introduction Cisplatin (cis-diamminedichloridoplatinum[II]) is one of the most commonly used antineoplastic drugs employed in treatment regimen for a broad range of human cancers. It has been used over the last decades in clinical oncology after Rosenberg first described its growthinhibiting effect on mammalian cells. 1-2 Cisplatin mediates its cytotoxic potential predominantly by covalently binding to nucleophilic positions in DNA, preferably to N7 in guanines or N3 in adenines. In a second step, such monoadducts are converted into intra- and inter-strand crosslinks with Pt-(GpG) representing the most abundant adduct type in cisplatin-modified DNA. 3-5

The rates of formation and the persistence of platination products in the nuclear DNA of

particular cells is determined by a number of factors including import / export mechanisms or intracellular sequestration of the drug, or efficient removal of adducts by various DNA damage repair mechanisms.

6-7

This holds true for both, tumor cells as well as for physiological cells in

healthy tissues. High accumulation of such adducts in particular subsets of cells are causative for the severe tissue-specific side effects of platinum drugs. To estimate the relative importance of all these mechanisms for the efficacy of platinum-based treatments and for the size of the “therapeutic window” analytical tools are necessary that allow the localization and assessment of specific platinum-DNA adducts, ideally at the level of individual cell nuclei and at clinically relevant drug doses.

8

Due to their high specificity and sensitivity, appropriate monoclonal

antibodies (Mab) immuno-assays are best suited for this purpose. The most frequently employed Mab to detect Pt-adducts in DNA is the IgG-type rat antibody R-C18 with a high affinity for the 2 ACS Paragon Plus Environment

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intrastrand crosslink Pt-(GG) that is already used for diagnostic purposes and molecular dosimetry studies in cancer therapy. In general, antibodies are classified into five groups (IgA, IgG, IgD, IgE and IgM), of which around 75 % of all antibodies in blood serum are IgGs. into four subtypes.

10-12

9

Furthermore, IgGs are divided

The R-C18 rat antibody investigated here belongs to the IgG2a subclass

and contains two identical light and heavy chains that are connected via two disulfide bridges (Fig. 1). The light chains harbor a variable (VL) and a constant (CL) region, while the heavy chains encompass a variable region (VH) and a constant region (CH1). Both form the Fab part (antigen-binding fragment) of the antibody that mediates its binding specificity. Furthermore, also two more constant regions (CH2 and CH3) are part of the heavy chain and build the Fc region (crystallizable fragment). Complex glycan structures are typically found in this region and are characteristic for most antibody species. 13-17 By reason of their remarkable binding, specificity antibodies are employed in various types of immunoassays to identify, localize, isolate or quantify distinct molecular structures including rare base modifications in DNA.

18

Therefore, they tend to be the most useful tool to

obtain a direct response from a target molecule like cisplatin.

19

In order to get reproducible

results, monoclonal antibodies are often used in bioanalytical sciences for diagnostic purposes. The monoclonal antibody R-C18 was established to detect cisplatin-induced Pt-adducts in genomic DNA and to allow quantitative analysis of drug-exposed biological or clinical samples. 20-21

It has been widely used in immuno-cytological assays, allowing dosimetric analyses at the

level of single cells.

8, 22-24

Although R-C18 has great potential to be used in clinical routine

diagnostics, a comprehensive characterization of this antibody is missing. The primary structure and modifications as well as the antigen specificity can be investigated by mass spectrometry (MS). MS based methods have been proven to be extremely useful in bioanalytical research, since the development of electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI).

25-26

Additionally, with the availability of tandem-MS experiments, proteomic

research passed through a rapid evolution in the last decade and protein sequencing became a standard technique.

27-28

However, studies of non-covalent interactions, like antigen-antibody-

complexes, still face intricate problems as they should be carried out under native conditions. Therefore, native MS methods are needed that allow the characterization of such systems under conditions that are as close as possible to the solution state. 29-30 In consequence, soft ionization 3 ACS Paragon Plus Environment


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methods and buffer systems should be used for analyses under native conditions. Moreover, the mass spectrometer has to be modified for low charges states and thus high m/z values. In such a system, an increased pressure in the first vacuum chamber of the mass spectrometer was shown to result in an increased transmission of large molecules, as the ions collide with remaining uncharged gas and solvent molecules and get decelerated (collisional cooling).

31-32

Moreover,

the transfer multipoles have to be modified so that higher m/z values can pass. 33-34 Here a comprehensive study is presented, in which the sequence, complex glycan structures and antibody-antigen-complexes of the cisplatin specific antibody R-C18 were characterized. As glycosylation represents 2-3 % of the total mass of IgG antibodies and has great influence on the antibody structure and antigen binding efficiency, the glycans at the position Asn293 in the CH2 region of the Fc part were further examined. These carbohydrates are typically N-glycans with a known core structure, only differing in the terminal monosaccharide residues.

35-38

Furthermore, not only the bare antibody, but also the antibody-antigen-complexes

were investigated with respect to specificity and binding conditions.

Materials & Methods Chemicals Enzymes and corresponding buffers were purchased from Promega, Fluka and New England Biolabs. Cisplatin, Urea and Tris(2-carboxyethyl)-phosphine hydrochloride were purchased from Sigma Aldrich. Other chemicals were supplied by Carl Roth. Monoclonal Antibody Purification The antibody was diluted in binding buffer (100 mM Na3PO4, 150 mM NaCl, pH 7.2) to a concentration of ~5 mg mL-1 and this solution was added to a Protein G spin column (Thermo Fisher Scientific). After 10 min of shaking, the solution was removed by centrifugation (1 min, 14,000 g) and the column was washed three times with 400 µL of binding buffer. Subsequently, the column was washed three times with 400 µL of elution buffer (100 mM glycine, pH 2.5) and the supernatants were collected in 40 µL of neutralization buffer (1 M Tris, pH 8.5) ESI-MS Parameter

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The antibody and the antibody-antigen-complexes were analyzed in the positive ion mode in concentrations of 1-2 mg.mL-1 in 200 mM ammonium acetate solution using a High-Mass QToF 2 mass spectrometer (Micromass Waters, modified by MS Vision). Typically, a capillary voltage of 1.0 kV was used. For the analyses of the antibody, the cone voltage was set to 99 V and the extractor voltage to 100 V. For the analyses of the antibody-antigen-complex, the cone and the extractor voltage were both decreased to 50 V. The collision energy was varied between 10 eV and 100 eV to increase desolvation of ions. Proteolytic digestions were assessed via nanoHPLC-ESI-FTICR-MS/MS. To do so, an Agilent 1100 nano-HPLC system was equipped with a Zorbax 300 SB C-18, 75 µm x 150 mm (particle size 3.5µm) column for separation. The flow rate was 0.35 µL.min-1 with a water/acetonitrile/formic acid eluent and a 60 min gradient (supplementary information). The HPLC was coupled to a NanoMate-ESI-Interface (Advion) and a Thermo Fisher LTQ FTICR Ultra. The MS parameters were 1.7 kV spray voltage, 200 °C capillary temperature, resolution 100,000. The three most abundant signals were isolated with ∆m/z 4 isolation width and fragmented with 35 % normalized CID energy. Only signals with a charge state of two or larger were taken into account for fragmentation. Data analysis was performed manually with an allowed mass deviation of lower than 5 ppm. DNA oligomers were analyzed in a concentration of 1 µM for single and 10 µM for double strands in 200 mM ammonium acetate solution with the High-Mass Q-ToF. The cone voltage was set to 50 V and the extractor voltage to 10 V. The collision energy was varied between 40 eV and 60 eV. PNGaseF and MALDI-MS For complete deglycosylation, 50 µg of the antibody were incubated with 1 µL PNGaseF (500 units) for 2 h at 37 °C. For MALDI-MS, the solution was desalted with cation exchanger beads (Amberlite, Merck KGaA). A 10 µM solution of the glycans was mixed with an equal volume of a 20 mg.mL-1 solution of 2,5-dihydroxybenzoic acid in 50:50 (v/v) water/acetonitrile. A LTQ Orbitrap XL (Thermo Fisher) with MALDI source was used for the analyses. Proteolytic digestion The antibody stock solution was diluted in 8 M urea solution to a final concentration of 1 mg.mL-1 and reduced with tris(2-carboxyehtyl)phosphine (50 mM in water) at a final concentration of 10 mM. Subsequently, the mixture was incubated with a two-fold excess of iodoacetamide for 1 h at 37 °C in the dark. The solution was split into two equal portions, of 5 ACS Paragon Plus Environment


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which one was deglycosylated with PNGaseF (5 µL, 2500 units). The PNGaseF treated samples were again split into six equal portions and each incubated with one enzyme (Chymotrypsin, GluC, LysC, LysN, Thermolysin, Trypsin) at a ratio of 1:100 each, in the corresponding enzyme buffer (supplementary information). All digests were incubated over night at 37°C, while the GluC treated sample was incubated at room temperature. All samples were analyzed via nanoHPLC-ESI-FTICR-MS/MS in 2 % formic acid solution. Mass spectrometric data analysis Peptide identification was achieved utilizing the Software PEAKS 8.0 (Bioinformatics Solutions).

The

system

was

set

to

the

specific

proteolytic

enzyme

used

and

carboamidomethylation was used as an additional fixed modification. A FASTA file was created that contained the Aldevron sequence, the sequences of all used enzymes, the sequence of human keratin and the sequence of the rat antibody (Uniprot Q5M842). With the de novo function of PEAKS, it is also possible to identify peptides that are not contained in the search database, but were detected and yielded suitable fragment spectra. The mass error was set to a maximum of 5 ppm in all cases. TBE Gels In order to investigate antibody-antigen-complex formation, native TBE gels were used. Silver staining was used to detect both antibody and antigen bands and SYBR Gold was used to stain DNA. A 6 % separation gel using 40 % acrylamide/bisacrylamidesolution (19:1) combined with a stacking gels was used. The stacking gel contained 675 µL of a 40 % acrylamide/bisacrylamide solution (37.5:1) and 575 µL of a 40 % acrylamide/bisacrylamide solution (19:1) per 10 mL. Before adding the sample, the gel was conditioned at 15 mA for 15 min. The sample solutions, containing both antibody and antigen in a molar ratio of 1:2 and a concentration of 0.5 mg.mL-1 of antibody, were incubated for 2 h at 37°C. Subsequently, 3.5 µL of this sample were mixed with 0.5 µL DNA loading buffer (10 mM Tris-HCl, 0.03 % bromophenol blue, 0.03 % xylene cyanol, 60 mM EDTA, 60 % glycerol, pH 7.6) and 3.5 µL water, followed by incubation at 37 °C for 10 min. Samples were loaded onto the gel and separation was carried out utilizing 25 mA. SYBR Gold staining was achieved by immersing the gel for 10 min in a 1:20,000 dilution of SYBR Gold in TBE buffer at room temperature and


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direct assessment of the fluorescence at 490/535 nm, followed by inversion of the obtained picture. ELISA The binding activity of the antibody was evaluated by ELISA. 96 well-plates were coated with platinated DNA. Therefore, cisplatin treated calf thymus DNA (ratio Pt:nucleotide 1:100) was sheared into approximately 300 base pair fragments using ultrasonication. For coating, 50 µL of the DNA solution (10 ng - 200 ng DNA) were added per well and allowed to dry overnight. After 5 min of irradiation with UV light (0.12 J), the plates were washed four times with water and once with PBS buffer. The antibody was diluted in PBS buffer and 50 µL were added per well, followed by incubation for 1 h at 37 °C. Subsequently, the supernatant was removed and the wells were rinsed four times with water and once with PBS buffer. The secondary antibody (goat-anti-rat mAb, Dianova, Germany, 0.6 mg.mL-1) was diluted (1:1,000) in a 1 % ovalbumin solution in PBS buffer. Each well was filled with 50 µL of this solution and the plate was incubated for 1 h at 37 °C. The supernatant was removed and the wells were rinsed four times with water. Finally, 100 µL of para-nitrophenyl phosphate solution (1 mg.mL-1) were added, followed by incubated for 30 min at 37 °C. The absorption was measured at 405 nm. Platination and hybridization of DNA oligomers For hybridization, equimolar amounts of the complementary 50-mer DNA-oligomers (BioTeZ Berlin-Buch GmbH, Berlin, Germany) were mixed in 200 mM ammonium acetate solution and heated to 95°C for 5 min. After slow cooling (30 min) to room temperature, the double stranded DNA was examined by TBE-PAGE. For platination, 0.5 mg cisplatin was dissolved in 1 mL water at 37°C. The cisplatin solution was then mixed with the DNA oligomer solution (100 µM in 200 mM ammonium acetate solution) in a 1:1 molar ratio and incubated over night at 37°C. Antibody-Antigen-Complex To form an antibody-antigen-complex, solutions of both were mixed in a 1:1 molar ratio (antibody 1-2 mg.mL-1, Pt-oligomers 50 µM, both in 200 mM ammonium acetate solution) and incubated for 2 h at 37°C. Afterwards, the complex was washed with 450 µL ammonium acetate solution utilizing centrifugal filters (Amicon Ultra, Merck Millipore, 3-100 kDa) at 7,000 g.



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Results Antibody structure and sequence The antibody was assessed by separation on reducing 2D-gels to visualize potential glycosylation sites and to determine apparent masses. The light chain was easily observed as a sharp spot with a mass around 25 kDa, whereas 50 kDa could be determined for the heavy chain (supplementary information). The heavy chain was not focused sharply, but gave a broad spot between pH 9.0 und pH 10.5, which indicates extensive post-translational modifications. To further investigate the antibody, native MS was used to determine a more exact mass. Thus, the antibody was deglycosylated with PNGaseF and assessed by nanoESI-MS and increasing the collision energy to lose non-covalently bound water molecules.

39

The most abundant species

was observed at 143,557.6±3.2 Da with some micro heterogeneities that stemmed from posttranslational modifications. Previously, the hypervariable part of the antibody and the light chain were sequenced by Aldevron (Freiburg, Germany) from the corresponding DNA. In order to verify these sequences, the light and the heavy chains were sequenced at the protein level. Therefore, the antibody was denatured in urea solution, reduced and the free thiol groups were alkylated with iodoacetamide. The antibody was then incubated separately with six different proteolytic enzymes before and after deglycosylation with PNGaseF. The latter was applied to increase the accessibility of proteases.

40

The constant region of the heavy chain was unraveled

on the basis of a hybrid sequence from a rat IgG2a antibody sequence (accession no. Q5M842), based on the approach by on Brüggemann et al..

41

By using this method, the sequence of the

antibody R-C18 was completely determined on the protein level with a sequence coverage of 100% (supplementary information). Accordingly, the theoretical mass was calculated to be 143,429.3 Da. This was in good agreement with the experimental mass determined by native MS (143,557.6 Da), while only a difference of around 130 Da was observed. This remaining difference can easily be explained by post-translational modifications that are not reflected by the theoretical mass, as several modifications have been detected (see below and supplementary information). Furthermore, incomplete desolvation also leads to determination of higher masses in native MS. Post Translational Modifications 8 ACS Paragon Plus Environment

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Extensive modifications were determined by gel electrophoresis showing a broad spot of the heavy chain (see supplementary information) and in native MS analyses of the complete antibody by micro heterogeneities within the antibody signals. In order to determine the glycosylation motifs, the glycans were released from the antibody with PNGaseF and analyzed by positive mode MALDI-MS experiments and detected as sodium adducts [M+Na]+. 42 In total, 15 different oligosaccharide species were found (Fig. 2). The m/z values differed less than 5 ppm from the calculated masses and the experiment was repeated four times. Besides various species of fucosylated and non-fucosylated N-glycans, different grades of galactosylation were observed. As previously shown, intensities can be directly compared to draw conclusions of the actual oligosaccharide distribution. 43-44 Therefore, the absolute intensities of the mass spectra were transformed into relative abundances of respective glycans (Fig. 3). The G0F glycosylation represents the most abundant signal with around 30 % of relative intensity, which is in good accordance to commonly known glycosylation patterns of monoclonal IgGs.

45-47

In general about 70 % of all saccharide structures were fucosylated and around 28 %

of the glycans held terminal galactose residue, whereby 90 % of these were only monogalactosylated. A bisecting GlcNAc unit, which is present as a third glycosylation arm at the mannose unit, was contained in 20 % of the structures. Within these experiments in the positive ion mode and also further experiments in the negative ion mode, no sialylated species were detected. To further investigate the glycosylation motifs and modification sites, glycopeptides were generated with the six proteases used for sequencing and analyzed with HPLC-ESI-MS/MS. In comparison to the MALDI-MS spectra, the G0(-2gn) and G0F(-2gn) species were not detected by this approach, but one sialylated peptide with the glycosylation motif G1FSia was clearly determined.

47

It is known, that sialic acid species are hardly detectable in MALDI without

derivatization and in addition the G1FSia glycosylation was of low abundance (under 1 %), as determined by HPLC-MS. All in all, 16 different glycosylation motifs at the typical position Asn293 were determined by combined MALDI-MS, ESI-MS and -MS/MS analyses. These results were again in good accordance with known glycan structures of rat IgG2a.

16, 46

Furthermore,

besides glycans, also other post translational modifications were detected (supplementary information). 9 ACS Paragon Plus Environment


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Antibody-Antigen-Complex To investigate the antigen binding of the antibody, oligonucleotides with known sequence were employed. The 1,2-Pt(GG) adduct is known to be the preferred binding motif for R-C18, 8 therefore, oligonucleotides treated with cisplatin were chosen as antigens. More precisely, two complementary single strand 50-mers were chosen as model system for the generation of antigens, with one strand containing two adjacent guanosines in the middle of the sequence (supplementary information). The purity of the single strands was verified by HPLC-UV and TBE-PAGE.

48

Furthermore, double strands were generated and assessed with nanoESI-Q-ToF-

MS. The single and double strands were subsequently treated with cisplatin. In ESI-MS spectra, signals for the unplatinated, mono platinated and double platinated double strands were found. Furthermore, both 50-mer single strands were observed as non-, singly and doubly platinated species (Fig. 4). Hence, it is clear that other platination species apart from 1,2-Pt(GG) were formed. Even though 1,2-Pt(GG) is preferred, former studies already determined other binding motifs for cisplatin.

3-5

To investigate the complex formed by the R-C18 antibody, non- and

singly platinated single and double strand DNA oligomers were incubated with the antibody and analyzed by native TBE-PAGE gels. To visualize DNA and DNA containing complexes on the gel, the intercalation dye SYBR Gold was used for staining, while both antibody and DNA were visualized by silver staining. For the unplatinated species, only signals for the DNA strands were detected, whereas no antibody bands were visible (Fig. 5A, lanes 1-3) in silver staining. For all platinated samples, DNAs were detected as single strands and the antibody-antigen bands were apparent (Fig. 5A, lanes 4-6). To verify this, SYBR Gold staining that only visualizes DNA was additionally used. Here, in the unplatinated reaction mixtures, only signals for the corresponding single and double strands were detected (Fig. 5B, lanes 1-3). In the platinated DNA cases, signals for the single and double strands were detected as well as the antibody-antigen complex bands (Fig. 5B, lanes 4-6), which showed the specificity of the antibody to cisplatin treated DNA oligomers. In a next step, the pH dependence of antigen binding was examined via enzymelinked immunosorbent assay (ELISA). For this purpose, the double stranded DNA as antigen and the antibody were incubated directly in ammonium acetate solutions with different pH values (4.0-7.0) and assessed by ELISA.

49

Additionally, the antibody-antigen-complex was formed in

PBS buffer and subsequently incubated in ammonium acetate solutions with different pH values (Fig. 6). 10 ACS Paragon Plus Environment

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In case the complex was formed in PBS buffer, the determined stability was constant at pH 4.5 and higher values. Only 77 % of activity were observed for the lowest pH value tested (pH 4.0). Here, the binding site seemed to be shielded by the antigen, so that denaturation by low pH values was prevented. Otherwise, if the complex was formed directly in ammonium acetate solution, the complex formation decreased to 20 % at pH 4.0. Comparable results were observed in an ammonium acetate solution of pH 7.0 and PBS buffer. The reason for this decrease was most likely denaturation of the antibody and therefore change of conformation of the paratope. 50 Furthermore, the antibody-antigen complex was analyzed with native ESI-MS in positive ion mode. Different collision energies were applied (up to 80 eV) in order to desolvate the complex (Fig. 5C). A charge series for the free antibody and for the 1:1 antibody-antigen complex was observed in high abundance. Moreover, also the 1:2 antibody-antigen complex was formed. Thus, no steric hindrance of the first bound antigen did influence the binding of a second antigen. Unplatinated single and double stranded DNA, and platinated single DNA strands were also incubated with the antibody as controls. No complex was found in the control experiments with unplatinated DNA, whereas antibody-antigen complexes were also found with both platinated single strands. Earlier experiments have shown that findings in the gas phase are also valid for the condensed phase. 51-52

Discussion In conclusion, the antibody R-C18 was comprehensively characterized in this study. Determination of the exact antibody mass was performed via PAGE and MS experiments. The antibody was completely sequenced at the DNA level, which was validated by digestion and MS/MS experiments on the protein level and alignment to known antibody sequences. The glycosylation decoration, as one of the most relevant characteristic structural property, was investigated and 16 different glycan motifs were identified. Finally, antibody-antigen-complexes were examined in detail. We could show that the pH has a great influence on complex formation, most likely due to denaturation or other changes of the binding site at low pH values. The antibody specificity was verified via incubation with platinated and non-platinated single and double strand DNA oligomers. While unplatinated species were not bound by the antibody, stable antibody-antigen-complexes were formed with platinated oligonucleotides. Here, binding 11 ACS Paragon Plus Environment


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of single and double stranded DNA was observed as well as simultaneous binding of up to two antigens. Furthermore, the antibody bound not only to 1,2-Pt(GG) motif, but also to other cisplatin binding motifs. In general, diagnostic applications and therapies can be supported rapidly and exactly by antibody based analyses. R-C18, as a first cisplatin-DNA binding antibody, shows great potential to further increase its applications by understanding and optimizing the antigen binding specificity with the help of modifications of the antibody sequence or its extensive modifications. This approach is promising, connecting structure related information with patient therapy. With the availability of the antibody sequence, further in detail investigations of the antibody-antigen binding have been enabled. This will lead to improved detection of cisplatin and its distribution in the human body in the future. Additionally, targeted modification (e.g. point mutations or amino acid alterations), to tune the antibody specificity for further more specific needs, have now been enabled. This would for instance also include to tailor the antibody to recognize other specific cisplatin binding sites in DNA.

Acknoweledgements We would like to thank Dr. Karola Lehmann from the Proteome Factory AG for 2D-gels. Conflict of interest The authors declare that they have no potential conflict of interest.

References 1. Rosenberg, B.; Vancamp, L.; Krigas, T., Inhibition of Cell Division in Escherichia Coli by Electrolysis Products from a Platinum Electrode. Nature 1965, 205 (4972), 698-+. 2. Rosenberg, B.; Vancamp, L.; Trosko, J. E.; Mansour, V. H., Platinum Compounds - a New Class of Potent Antitumour Agents. Nature 1969, 222 (5191), 385-+. 3. Fichtinger-Schepman, A. M.; van der Veer, J. L.; den Hartog, J. H.; Lohman, P. H.; Reedijk, J., Adducts of the antitumor drug cis-diamminedichloroplatinum(II) with DNA: formation, identification, and quantitation. Biochemistry-Us 1985, 24 (3), 707-13. 4. Rudd, G. N.; Hartley, J. A.; Souhami, R. L., Persistence of cisplatin-induced DNA interstrand crosslinking in peripheral blood mononuclear cells from elderly and young individuals. Cancer Chemother Pharmacol 1995, 35 (4), 323-6. 5. Poklar, N.; Pilch, D. S.; Lippard, S. J.; Redding, E. A.; Dunham, S. U.; Breslauer, K. J., Influence of cisplatin intrastrand crosslinking on the conformation, thermal stability, and energetics of a 20-mer DNA duplex. Proc Natl Acad Sci U S A 1996, 93 (15), 7606-11.


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24. Nel, I.; Gauler, T. C.; Eberhardt, W. E.; Nickel, A. C.; Schuler, M.; Thomale, J.; Hoffmann, A. C., Formation and repair kinetics of Pt-(GpG) DNA adducts in extracted circulating tumour cells and response to platinum treatment. Br J Cancer 2013, 109 (5), 1223-9. 25. Karas, M.; Hillenkamp, F., Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem 1988, 60 (20), 2299-301. 26. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray ionization for mass spectrometry of large biomolecules. Science 1989, 246 (4926), 64-71. 27. Wells, J. M.; McLuckey, S. A., Collision-induced dissociation (CID) of peptides and proteins. Methods Enzymol 2005, 402, 148-85. 28. Mechref, Y.; Novotny, M. V.; Krishnan, C., Structural characterization of oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry. Anal Chem 2003, 75 (18), 4895-4903. 29. Heck, A. J. R., Native mass spectrometry: a bridge between interactomics and structural biology. Nat Methods 2008, 5 (11), 927-933. 30. Loo, J. A., Electrospray ionization mass spectrometry: a technology for studying noncovalent macromolecular complexes. Int J Mass Spectrom 2000, 200 (1-3), 175-186. 31. Schmidt, A.; Bahr, U.; Karas, M., Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS. Anal Chem 2001, 73 (24), 6040-6046. 32. Chernushevich, I. V.; Thomson, B. A., Collisional cooling of large ions in electrospray mass spectrometry. Anal Chem 2004, 76 (6), 1754-1760. 33. Sobott, F.; Hernandez, H.; McCammon, M. G.; Tito, M. A.; Robinson, C. V., A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. Anal Chem 2002, 74 (6), 1402-1407. 34. van den Heuvel, R. H.; van Duijn, E.; Mazon, H.; Synowsky, S. A.; Lorenzen, K.; Versluis, C.; Brouns, S. J.; Langridge, D.; van der Oost, J.; Hoyes, J.; Heck, A. J., Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry. Anal Chem 2006, 78 (21), 7473-83. 35. Wright, A.; Morrison, S. L., Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol 1997, 15 (1), 26-32. 36. Beck, A.; Wagner-Rousset, E.; Bussat, M. C.; Lokteff, M.; Klinguer-Hamour, C.; Haeuw, J. F.; Goetsch, L.; Wurch, T.; Van Dorsselaer, A.; Corvaia, N., Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. Curr Pharm Biotechnol 2008, 9 (6), 482-501. 37. Guddat, L. W.; Herron, J. N.; Edmundson, A. B., Three-dimensional structure of a human immunoglobulin with a hinge deletion. Proc Natl Acad Sci U S A 1993, 90 (9), 4271-5. 38. Varki, A., Loss of N-glycolylneuraminic acid in humans: Mechanisms, consequences, and implications for hominid evolution. Am J Phys Anthropol 2001, Suppl 33, 54-69. 39. Liu, J.; Nguyen, M. D.; Andya, J. D.; Shire, S. J., Reversible self-association increases the viscosity of a concentrated monoclonal antibody in aqueous solution. J Pharm Sci 2005, 94 (9), 1928-40. 40. K. Hooper, M. R., M. Urh, S. Savaliev, C. Hosfield, G. Kobs, M. Ford, R. Jones, R. Amunugama, D. Allen, R. Brazas, Alternative Enzymes Lead to Improvements in Sequence Coverage and PTM Analysis. Journal of Biomolecular Techniques 2013, 24, 52. 41. Bruggemann, M., Evolution of the rat immunoglobulin gamma heavy-chain gene family. Gene 1988, 74 (2), 473-82. 42. Morelle, W.; Michalski, J. C., Analysis of protein glycosylation by mass spectrometry. Nat Protoc 2007, 2 (7), 1585-602. 43. Signor, L.; Erba, E. B., Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa. Jove-J Vis Exp 2013, (79).


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44. March, R. E.; Todd, J. F. J., Quadrupole Ion Trap Mass Spectrometry, 2nd Edition. Chem Anal Series Mon 2005, 165, 1-351. 45. Raju, T. S.; Briggs, J. B.; Borge, S. M.; Jones, A. J. S., Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 2000, 10 (5), 477-486. 46. Zhang, Z. Q.; Pan, H.; Chen, X. Y., Mass Spectrometry for Structural Characterization of Therapeutic Antibodies. Mass Spectrom Rev 2009, 28 (1), 147-176. 47. Jefferis, R., Glycosylation as a strategy to improve antibody-based therapeutics. Nat Rev Drug Discov 2009, 8 (3), 226-234. 48. Brody, J. R.; Kern, S. E., History and principles of conductive media for standard DNA electrophoresis. Anal Biochem 2004, 333 (1), 1-13. 49. Dewerth, M. A.; Eicher, C.; Ellerkamp, V.; Kirchner, B.; Warmann, S.; Fuchs, J.; Armeanu-Ebinger, S., Effect of sorafenib in combination with cytostatic agents on hepatoblastoma in vitro and in vivo. Cancer Res 2012, 72. 50. Van Oss, C. J., Hydrophobic, hydrophilic and other interactions in epitope-paratope binding. Mol Immunol 1995, 32 (3), 199-211. 51. Atmanene, C.; Wagner-Rousset, E.; Malissard, M.; Chol, B.; Robert, A.; Corvaia, N.; Van Dorsselaer, A.; Beck, A.; Sanglier-Cianferani, S., Extending mass spectrometry contribution to therapeutic monoclonal antibody lead optimization: characterization of immune complexes using noncovalent ESI-MS. Anal Chem 2009, 81 (15), 6364-73. 52. Oda, M.; Uchiyama, S.; Noda, M.; Nishi, Y.; Koga, M.; Mayanagi, K.; Robinson, C. V.; Fukui, K.; Kobayashi, Y.; Morikawa, K.; Azuma, T., Effects of antibody affinity and antigen valence on molecular forms of immune complexes. Mol Immunol 2009, 47 (2-3), 357-64.



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Figure legends Fig 1. Schematic structure of an IgG2a antibody. Fig 2. MALDI-MS spectra of the PNGaseF cleaved glycans in DHB matrix.47

Fig 3. Relative abundances of the 15 antibody glycan motifs analyzed by MALDI-MS. The given errors represent the standard deviation of four independent samples of the deglycosylated antibody. Non-fucosylated structures are marked in gray, fucosylated in black-white.

Fig 4. ESI-MS spectra of the platinated single strand 1 that includes the GG-motif (left), the platinated single strand 2 (middle) and the platinated double strand 12 (right). Both single strands bound cisplatin, also single strand 2 that did not contain a GG motif. The non platinated species is marked with 0x Pt, the singly platinated with 1x Pt and the doubly platinated with 2x Pt. Fig 5. Gel electrophoretic separation and mass spectrometric analysis of reaction mixtures to investigate antibody-antigen complex formation. A – native TBE gel with silver staining for antibody and DNA detection, B – native TBE gel with SYBR Gold staining for DNA. Reaction mixtures: Lane 1 – antibody with DNA single strand 1 not containing cisplatin, Lane 2 – antibody with DNA single strand 2 not containing cisplatin, Lane 3 – antibody with DNA double strand 12 not containing cisplatin, marker lane, Lane 4 – antibody with DNA single strand 1 containing cisplatin, Lane 5 – antibody with DNA single strand 2 containing cisplatin, Lane 6 – antibody with DNA double strand 12 containing cisplatin, C - nanoESI-Q-ToF-MS spectrum of the antibody-antigen complex with the platinated double strand (1 mg.mL-1 in 200 mM NH4OAc solution, DNA/cisplatin 1:1). The signals for the free antibody are marked with ○, the signals for the 1:1 complex with ✱ and the 1:2 complex with ✜.

Fig 6. Antibody-antigen formation based on UV absorption in ELISA in dependence of the pH value in which the antibody and the antigen were incubated. The darker bars mark experiments where the samples were incubated directly in NH4OAc solution of different pH values and the lighter bars show incubation in PBS buffer with subsequent post incubation in NH4OAc solution. 16 ACS Paragon Plus Environment

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Graphical Abstract 32x11mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Fig 1. Schematic structure of an IgG2a antibody. 60x44mm (300 x 300 DPI)


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MALDI-MS spectra of the PNGaseF cleaved glycans in DHB matrix.47 82x38mm (300 x 300 DPI)



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. Relative abundances of the 15 antibody glycan motifs analyzed by MALDI-MS. The given errors represent the standard deviation of four independent samples of the deglycosylated antibody. Non-fucosylated structures are marked in gray, fucosylated in black-white. 80x50mm (300 x 300 DPI)


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ESI-MS spectra of the platinated single strand 1 that includes the GG-motif (left), the platinated single strand 2 (middle) and the platinated double strand 12 (right). Both single strands bound cisplatin, also single strand 2 that did not contain a GG motif. The non platinated species is marked with 0x Pt, the singly platinated with 1x Pt and the doubly platinated with 2x Pt. 53x16mm (300 x 300 DPI)



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Gel electrophoretic separation and mass spectrometric analysis of reaction mixtures to investigate antibodyantigen complex formation. A – native TBE gel with silver staining for antibody and DNA detection, B – native TBE gel with SYBR Gold staining for DNA. Reaction mixtures: Lane 1 – antibody with DNA single strand 1 not containing cisplatin, Lane 2 – antibody with DNA single strand 2 not containing cisplatin, Lane 3 – antibody with DNA double strand 12 not containing cisplatin, marker lane, Lane 4 – antibody with DNA single strand 1 containing cisplatin, Lane 5 – antibody with DNA single strand 2 containing cisplatin, Lane 6 – antibody with DNA double strand 12 containing cisplatin, C - nanoESI-Q-ToF-MS spectrum of the antibodyantigen complex with the platinated double strand (1 mg.mL-1 in 200 mM NH4OAc solution, DNA/cisplatin 1:1). The signals for the free antibody are marked with ○, the signals for the 1:1 complex with ✱ and the 1:2 complex with ✜. 165x101mm (300 x 300 DPI)


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Antibody-antigen formation based on UV absorption in ELISA in dependence of the pH value in which the antibody and the antigen were incubated. The darker bars mark experiments where the samples were incubated directly in NH4OAc solution of different pH values and the lighter bars show incubation in PBS buffer with subsequent post incubation in NH4OAc solution. 70x38mm (300 x 300 DPI)


specific monoclonal antibody

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