An anti-caffeine antibody-oligonucleotide conjugate for DNA-directed

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An anti-caffeine antibody-oligonucleotide conjugate for DNAdirected immobilization in environmental immunoarrays Ana Margarida Carvalho, Cinthya Yamila Véliz Montes, Rudolf J. Schneider, and Annemieke Madder Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01347 • Publication Date (Web): 08 Aug 2018 Downloaded from http://pubs.acs.org on August 11, 2018

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An anti-caffeine antibodyoligonucleotide conjugate for DNA-directed immobilization in environmental immunoarrays Ana Margarida Carvalho, †,‡ Cinthya Véliz Montes, ‡ Rudolf J. Schneider,‡* Annemieke Madder†* †

Ghent University, Faculty of Sciences, Department of Organic and Macromolecular Chemistry,

Organic and Biomimetic Chemistry Research Group, Krijgslaan 281 (S4), 9000 Ghent, Belgium ‡

BAM Federal Institute for Materials Research and Testing, Department of Analytical

Chemistry; Reference Materials, Richard-Willstätter-Str. 11, D-12489 Berlin, Germany ABSTRACT: The development of fast and cheap high-throughput platforms for the detection of environmental contaminants is of particular importance to understand human-related impact on the environment. The application of DNA-directed Immobilization (DDI) of IgG molecules is currently limited to the clinical diagnostics scenario, possibly due to the high costs of production of such addressable platforms. We here describe the efficient and specific hybridization of an antibodyoligonucleotide conjugate to a short 12-mer capture probe. The specific antibody used is a monoclonal antibody against caffeine, a stimulant and important anthropogenic marker. With this work we hope to contribute to broadening the application potential of DDI to environmental markers in order to develop cheaper and more stable high-throughput screening platforms for standard routine analysis of pollutants in a variety of complex matrices. INTRODUCTION

Monoclonal antibodies are key components of immunoassays and the versatility of their application results from the binding specificity of these proteins. They can be raised against virtually any antigen of interest1, binding their epitopes with high affinity2 even when immersed

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in complex matrices3. Several formats of immunoassays are indispensable tools in disease-state profiling4,5, biosensing6,7, as well as food analysis and drug screening8–10. Among these formats, immuno-capture in lateral flow devices, Western blot and enzyme-linked immunosorbent assays (ELISA) have been around for decades, and remain the state-of-the-art in routine technologies. Compared to classical immunoassay formats, antibody arrays (immunoarrays) offer additional advantages in terms of miniaturization, speed of analysis, high multiplexing potential as well as low reagent consumption and high sensitivity2,11. Despite overcoming formidable challenges, antibody microarrays still fall short in routine diagnostics and did not massively hit analytical markets12,13. The main reason can be found in the low reproducibility of immunoarray production, impacting the quantification readout.14–17. Direct antibody immobilization on an array surface poses real challenges regarding storage stability of the immunoarray13 and often results in a low proportion of antibodies with accessible and functional binding sites. The interaction antibody/surface is still under scrutiny as few experimental techniques are available to provide protein immobilization information at atomiclevel resolution.12,15 Molecular simulation has emerged as the primary method to study protein/surface interactions. For example, Grawe and Knotts have studied different positions within the three dimensional structure of an IgG-type antibody tethered to both hydrophilic and hydrophobic surfaces15. The results showed that antibodies docked to hydrophilic surfaces were in general more stable15. Surface hydrophilicity has indeed been reported to be an important contributor to antibody adsorption, based on the evidence of more homogeneous spots and better stability during storage16. Gerdtsson and co-workers conducted a comprehensive study of the surface impact on antibody array performance.13 They compared twenty-two commercially available solid platforms for immunoarray technology in terms of spot reproducibility, signal intensity, overall sensitivity and non-specific protein binding. They showed that these parameters are strongly dependent on the chemical nature of the solid support13. In contrast to antibody array technology, DNA microarrays are being routinely employed for expression profiling and genotyping.17–21 The primary reason is that DNA is a highly stable molecule which, unlike an antibody, better tolerates covalent immobilization under reactive surfaces without losing its probing ability22–24. Also, oligonucleotides bearing chemical


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modifications for the direct attachment to surfaces are readily available, either via phosphoramidite chemistry25 or by in situ sequence printing using inkjet DNA synthesizers.26 DNA-directed immobilization (DDI) of antibodies, introduced over 20 years ago22, takes advantage of surface-bound capture oligonucleotides to selectively bind proteins tagged with complementary oligomers. Owing to the specificity of Watson-Crick base pairing, different DNA-tagged antibodies can be simultaneously immobilized27. Such a mild immobilization technique provides the antibodies a “soft landing” on the slide thus minimizing their denaturation. A comparative performance study was conducted by Washburn et al. to actually distinguish the antigen binding capacity of an anti-PSA-5A6 antibody immobilized either by direct covalent attachment or through DDI, under similar conditions28. The linear response for antigen binding was found to be 2.5 times larger when an antibody-DNA conjugate was used, in comparison to the same antibody covalently tethered to the surface28. Despite the fact that the mechanism for such a signal enhancement is not completely understood, it was proposed that a DNA functionalized surface is sufficiently hydrophilic to minimize antibody damage28. In addition, DNA might provide a more flexible linkage to favor a better orientation for antigen capture, as opposed to a shorter and more rigid covalent molecule28. Finally, DDI allows for a controlled addressing of the antibody on the slide, which enables positioning of the antibodies, e.g. by dipping the slide in a mix of different antibody oligos, and facilitates microarray readout. One can argue that, despite the advantages brought by DDI, there is an added source of expense and complexity by introducing a coupling step between the antibody and the oligonucleotide sequence. Conjugation as well as characterization of antibody-DNA constructs are difficult to achieve and, according to a recent review, represent itself, an independent field of pure and applied research27. Recently, we have contributed to this area by proposing a fast and cheap method to both produce and routinely characterize antibody-DNA conjugates29. Making use of similar synthetic and characterization approaches29, herein we move one step ahead. In this work a monoclonal antibody against caffeine, a stimulant and environmentally relevant contaminant30–32, was addressed on a microarray slide using 12-mer oligonucleotides as capture sequences. This probe size is well below the lengths typically reported in DDI applications (20-mer or higher)28,29,33, hence decreasing the overall cost of production of DNAbased antibody planar arrays.


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EXPERIMENTAL SECTION Instrumentation. An A10 Milli-Q water purification system from Millipore was used to obtain ultrapure reagent water for the preparation of buffers and dilutions. Gel filtration columns (Zeba™Spin, Thermo Fisher Scientific) were used in all purification and desalting steps. Incubation steps were performed on a Titramax 101 plate shaker (Heidolph). Absorbances of nucleic acids (λmax=260 nm), anti-caffeine mAb and anti-caffeine-mAb conjugates (λmax=280 nm) were determined on a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Gels for SDS-PAGE immunoanalysis were cast in a Mini-Vertical Unit (C.B.S. Inc.) and run by a Consort EV202 power supply (Sigma Aldrich). MALDI-ToF-MS spectra were acquired by an Autoflex III instrument operated with a FlexImaging 3.0 software (Bruker Daltonics). Origin version 8.5 was used for correcting the baseline, peak shape smoothing and fitting with a Gaussian distribution. ELISA washing steps were performed on an automatic 96-channel plate washer (ELx405 Select BioTek Instruments). The optical density (OD) was read on a microplate reader SpectraMax Plus384 (Molecular Devices) controlled by SoftMax Pro software version 5.2 (Molecular Devices). Microarray printing was performed using a BioOdyssey Calligrapher MiniArrayer (Bio-Rad) on transparent covalently modified streptavidin (STV) 3D glass slides (25 cm x 75 cm x 1 mm) (PolyAn). The glass slide was scanned using an Axon GenePix 4300A reader controlled by GenePix Pro 7.1 software. The laser power and photomultiplier tube (PMT) voltage were set to 60% and 400 V respectively.

Chemicals. Buffer salts and solvents were obtained from Sigma Aldrich in the highest purity grade. The bifunctional cross-linker succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC) and 3,3',5,5'-tetramethylbenzidine (TMB) substrate for ELISA were purchased from Thermo Fisher Scientific. Anti-caffeine mAb (1.23 mg/mL, mouse IgG 2b) was purchased from USBiological. Trastuzumab (Roche/Genentech) was used for mass calibration purposes prior to MALDI-ToF-MS analysis. Polyclonal anti-mouse IgG antibody (R12569, 2.2 mg/mL) was purchased from Acris Antibodies GmbH. A goat anti-mouse IgG labelled with Alexa Fluor 647 was purchased from Invitrogen (RJ243424, 2.0 mg/mL) and used for microarray detection of the immobilized conjugate. Casein used for blocking, molecular weight protein marker and sinapinic acid matrix for MALDI-ToF-MS were purchased from Protea


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Biosciences. Capture ON probes (12 bases) were synthesized and characterized as previously described24. The tagging DNA sequence to address the anti-caffeine antibody was purchased from Integrated DNA Technologies (IDT), already purified by HPLC. The information regarding sequence, molecular weight, modifications and stock solution concentrations of the oligonucleotides employed in this work are summarized in Table 1. Table 1. Oligonucleotide (ON) sequences and additional information. Sequence (5’3’)

Size (mer)

Molecular weight (MW, Da)

5’modification

X modification

Role

ON.A

TTTTTTTTTT GCACGCCGTCAG

22

6868.4

thiol modifier (C6-S-S-)

none

Tag sequence (conjugated to anti-caffeine mAb

ON.B

CTG ACG GXG TGC

12

4118.8

biotin

furan containing nucleoside (*)

ON.C

CAC AGC CXC TCG

12

3961.9

biotin

furan containing nucleoside (*)

Capture probe (positive control) Noncomplementary strand (negative control)

(*) The capture DNA sequences were available from previous work.24

Anti-caffeine mAb-DNA conjugate synthesis and characterization. The synthesis and characterization methodologies were similar to the ones described in previous work29 with minor alterations. The ELISA protocol was performed in a direct sandwich assay format as previously described34. The details of conjugation, SDS-PAGE, MALDI-ToF-MS and ELISA protocols can be found in Supporting Information. DNA capture probes – spotting and immobilization. Prior to immobilization, the streptavidin (STV) chips were washed with ultrapure water and gently dried under a gentle stream of argon. The solutions of biotinylated complementary oligonucleotide probes were prepared by diluting the stock solutions of the complementary oligonucleotide sequences to 2 µM and 20 µM (150 µL) in PBS pH 7.6. The solution of the non-complementary sequence was diluted to 20 µM. The three DNA solutions were loaded in three independent spotter channels (100 µL) in 8 replicates. The slide was kept at 4ºC overnight to maximize biotin-STV complex formation. A solution of PBS pH 7.6 and 0.05% Tween (PBST) was used to wash the slides and remove unreacted sequences. A casein solution supplemented with biotin (1 mg/mL) was then used to block the


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slide for 1h at RT. The slide was again washed using PBST followed by rinsing with ultrapure water and dried with a gentle argon flow.

Conjugate hybridization and detection. Two dilutions of the conjugate were tested in each sub-array, respectively 1:5 and 1:50. 50 µL of each dilution were deposited in the correspondent sub-array grid and incubated under gentle shaking (450 rpm) for 2 h at room temperature to allow hybridization. PBST solution followed by ultrapure water was used to respectively wash and rinse the slide, and it was allowed to air dry. The goat anti-mouse antibody labeled with Alexa 647 was diluted to 0.6 mg/mL and incubated for 2 h at room temperature under gentle shaking, and the slide was again washed with PBST and water and dried and assembled in the microarray reader.

RESULTS AND DISCUSSION Oligonucleotide-antibody conjugation. The conjugation of short oligonucleotides to a monoclonal antibody was performed by first activating the caffeine mAb with the commercially available bifunctional cross-linker SMCC. This cross-linker features two different activated groups, which can rapidly and selectively react with primary amines and sulfhydryl groups, to form amide and thioether bonds, respectively. It further holds the advantage of being cheaper than its analogous sulfonated derivative (available as sodium salt) (sulfo-SMCC). In comparison to antibody-oligonucleotide commercial conjugation kits (e.g. from SoluLinK) it also brings the advantage that only the antibody needs activation. The disadvantage of using this conjugation strategy is the random functionalization of the surface-exposed lysines of the antibody, which results in heterogeneous conjugate mixtures. The oligonucleotide sequences were purchased with a 5’-thiol termination to allow for covalent attachment to the maleimide-activated antibody. The exact details of the conjugation can be found in the supporting information. The conjugate was purified using a 40 kDa pore size gel filtration column to remove the excess, non-conjugated DNA. The concentration of the final conjugate solution was estimated by spectrophotometry at 280 nm to 0.65 mg/mL (± 0.04) mg/mL. This value represents the reference concentration for the following characterization experiments, but it is important to highlight that it accounts for both DNA-functionalized as well as non-functionalized antibodies.


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SDS-PAGE analysis of the conjugates. Both native anti-caffeine mAb and its DNA-conjugate were loaded on a 4-10% polyacrylamide handcast gel. Denaturing conditions are employed to allow for an efficient electrophoretic separation; however, heating of the samples was avoided, and the gel was run at 10ºC to minimize the risk of proteolysis. The exact details on gel and sample preparation, running conditions and staining can be found in SI. A picture of the silverstained gel is shown in Figure 1. Lane 2 shows the native caffeine mAb loaded under mildly denaturing and non-reducing conditions. Despite the temperature control and loading of the antibody samples at room temperature, 4 bands are present showing a tendency for this antibody to fragment. This fragmentation pattern has been observed and reported earlier for the same mAb isotype, IgG2b, as employed here.35 The complete mAb can be identified at 150 kDa, a band slightly below corresponds to the F(ab’)2 fragment (~120 kDa)35, and a band at approximately 100 kDa represents the Fab/c fragment35. The F(ab’)2 fragment is composed of the two Fab binding sites and the upper portion of the Fc-hinge. Tris(2-carboxyethyl)phosphine (TCEP) was employed to separate the heavy chain (Hc) and light chain (Lc) and improve the band resolution, as we have previously shown MW (kDa) 1 250_

150_

2

3

4

Caffeine IgG2b-DNA conjugate

100_ 75_

O NH

50_

N O

O S

Caffeine IgG2b Heavy chain (Hc)

n

37_ 25_

Caffeine IgG2b Light chain (Lc)

Figure 1. SDS-PAGE analysis of mAb DNA conjugates in a 4-10% discontinuous gel with silver stain band development. Lane 1: Molecular weight ladder (MW in kDa); Lane 2: Native caffeine antibody (no modification, prior to conjugation) Lane 3: TCEP reduced native caffeine mAb (prior to conjugation 1mg/mL). Lane 4: Caffeine mAb-DNA conjugate.


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that it is not possible to clearly identify the conjugates under non-reducing conditions.29 Upon treatment with TCEP this fragment is cleaved into another fragment composed of two light chains (LC) (25 kDa) and one partial heavy chain (HC) identified at 37.5 kDa (Figure 1, lane 2). The Fab/c fragment (Figure 1, lane 2, 100 kDa) is formed by an intact Fab domain and the Fc region and, after reduction, it is composed by only one LC and one HC (50 kDa)35. The mAbDNA conjugate was loaded in lane 4 after treatment with TCEP. Three slower migrating bands are clearly stained above the HC molecular weight region, which are not initially present in the native antibody (compare lanes 3 and 4). This indicates the covalent formation of the mAb-DNA conjugates and at least 3 migrating species can be identified. Based on the intensity of the stained bands (each band is approximately 10% of the maximum intensity), we estimate the reaction yield to be between 30%-50%. The resolving power of the gel at high MW is low. Therefore MALDI-ToF-MS was used to achieve a better estimation of the degree-of-labeling (DOL).

MALDI-ToF-MS. The average number of DNA molecules introduced per mAb molecule was estimated by spotting the samples in a sinapinic acid matrix in sandwich spotting mode as it provides high reproducibility for co-crystallization and sample ionization. Figure 2 illustrates the typical ionization pattern of a pure mAb. The two more intense peaks correspond to the protonated, singly charged species [M+H]+ and to the doubly charged species [M+2H]2+. The less intense peak corresponds to the dimer of the heavy chain in its triple-charged state, [2M+3H]3+.36 To estimate the parent molecular mass of anti-caffeine mAb, all peaks in the spectrum in Figure 2 were mass calibrated, divided by the mass factor and multiplied by the appropriate charge factor and averaged to yield an average mass and a corresponding standard deviation (SD). Table 2 summarizes the MS data for the measured and calculated molecular weight (MW) of the anti-caffeine mAb.


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Figure 2. Native caffeine mAb MALDI-ToF-MS profile showing the signals from the singly charged species (150 kDa), and doubly charged species (75 kDa).

Table 2. Parent Masses from peak signals of different mAb species used for the calculation of the average mass of the native anti-caffeine mAb. Standard deviations (σ) were calculated using a Gaussian fit function (σ =w/2) and propagated into the SD of the average MW. Signal / Charge

Mass factor

Charge factor

Parent mass

[M+2H]2+

75782.1637

1

2

151564.327

[M+H]+

151111.111

1

1

151111.111

[2M+3H]3+

101622.807

2

3

152434.210

Average MW ± SD

σ (m/z) 1.37E+03

151703 ± 672

3.34E+03 1.02E+05

Figure 3 shows the MALDI-ToF-MS spectrum obtained for the caffeine mAb conjugated to the DNA oligomer. The general trend observed is a broadening of the Gaussian distribution relatively to the native mAb (Figure 2). This is a typical feature for mAbs conjugated via random attachment to lysine side chains due to the neutralization of the charges upon conjugation with negatively charged DNA.36


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In addition to the signals of the singly and doubly charged species, it is also possible to identify the formation of two extra peaks at an m/z of 102 kDa and 127 kDa and relative intensities of 60 % and 34 % respectively, which were previously observed in SDS-PAGE (Figure 1, lane 2). These peaks can be attributed to the higher tendency of IgG2b mAb to fragment under MALDI conditions in comparison to IgG2a and IgG1 mouse isotypes.35 The first fragment observed is Fab/c (m/z 102 kDa, Figure 3). The tendency for cleavage of only one Fab domain was attributed to the asymmetric glycosylation of IgG2b, at the NH2-terminal side of the inter-heavy chain disulfide in the hinge region.35 The glycosylated site offers a better protection from enzymatic cleavage, whereas the non-glycosylated site was more prone to fragment, yielding a Fab/c fragment.35 The signal at 127 kDa presumably results from F(ab’)2 which is formed by the two Fab domains and a portion of the Fc region. Figure 3 shows the extended MS spectrum (65 to 200 kDa) obtained for the DNA conjugate of the anti-caffeine mAb. The resolution of the mass spectra is decreased due to the presence of non-conjugated mAb that ionizes preferentially.

Figure 3. MALDI-ToF-MS spectrum for caffeine mAb conjugated to a DNA sequence of 6968 Da.

For this reason, the m/z signals obtained were fitted individually using a Gaussian distribution to statistically reveal overlapping peaks. Results of this iteration are shown in Figure 4.


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Table 3 summarizes this information in terms of the peak distribution centers, mass shifts and degrees of labeling (DOL). x0 is defined as the center of the peak corresponding to the native anti-caffeine mAb signal. The difference between the m/z peak center of the conjugate (xc) and the peak center for the unconjugated mAb (x0) was calculated and represents the molecular weight shift, relatively to the parent signal. This value is then divided by the effective molecular weight of the oligonucleotide employed yielding the DOL distribution. The associated error was derived by propagating the uncertainty using the standard deviation (σ) from the peak centroid of a Gaussian curve (Table 2) previously obtained for the anti-caffeine antibody. The Gaussian fit to the peaks reveals a DOL between 1 and 4 oligonucleotide molecules per anti-caffeine mAb for the single and double charge (Figure 4D and Figure 4A, respectively), and a maximum DOL of 2 oligonucleotides per each antibody fragment Fab/c and F(ab’)2 (4B and 4C). It is important to highlight that the MS spectra depicted are complementary to the SDS-PAGE analysis. After SDS-PAGE we observe a reduced intensity for the antibody pattern in the lower molecular weight region. This technique is useful to have an idea of the degree of success of the conjugation reactions. The MALDI-ToF-MS performed according to the procedure described, in contrast, enables for an accurate prediction of the conjugate mass and the DOL distribution.

Figure 4. Gaussian peak fitting for individual m/z signals obtained using A) signal from the doubly charged species [M+2H]2+; B) Fab/c antibody fragment; C) F(ab’)2 fragment; D) signal from the singly charged species, [M+H] +. ACS Paragon Plus Environment

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Table 3. Interpretation of the Gaussian distribution shown in Figure 4 and determination of the number of oligonucleotide sequences attached to the anti-caffeine mAb.

Parent signal center (x0) [M+2H]2+

Mass shift (xc-x0)

DOL (*)

Error DOL

77658.30

2562.89

0.7

0.3

87872.95

12777.54

3.7

0.3

106104.98

4366.70

0.6

0.3

113723.88

11985.60

1.7

0.3

131745.61

4709.47

0.7

0.4

137583.01

10546.88

1.5

0.4

156630.65

5552.47

0.8

0.7

175590.44

24512.27

3.6

0.7

180114.88

29036.70

4.2

0.7

75095.41

Fab/c [M+H]+

101738.28

F(ab’)2

127036.13

[M+H]+

Conjugate peak center (xc)

151078.17

(*) Molecular weight of DNA sequence attached to anti-caffeine mAb is 6868.4 Da (ON.A).

ELISA evaluation of the binding capacity of the anti-caffeine mAb-DNA conjugates. Although remarkably stable under physiological conditions, the peptide linkage in antibodies is prone to degradation due to specific amino acid side chains, flexibility of the local structure, and reaction conditions.37 During the MALDI-ToF-MS analysis in the previous section, the formation of two antibody fragments was observed when analyzing the reaction mixture after DNA conjugation. The fragment at 100 kDa (Fab/c) is formed by only one antigen binding site and the Fc region, thus one Fab domain was lost during analysis of the conjugates. On the other hand, F(ab’)2 fragments contain the two binding sites, but the Fc domain is partially lost. It is therefore important to verify that this fragmentation only occurs because of the MALDI-ToF-MS analysis and does not affect the binding capacity of this antibody. The ELISA protocol for the anti-caffeine mAb has been extensively optimized and employed.34 We performed it to compare the binding activity of the DNA-conjugated antibody to the native anti-caffeine antibody. Results are shown in Figure 5. The assay was performed in duplicate and the raw OD values and SD results are presented in the supporting information.


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The conjugation reaction did not reach completion as can be derived from PAGE analysis, and, therefore, the results shown in this section cannot be directly correlated to the conjugate binding activity, but are rather an indication of the impact of the conjugation conditions and potential partial fragmentation on the final antibody activity. The signal of the native anti-caffeine antibody was normalized and represents the maximum affinity (average absolute OD of 2.54). For the anti-caffeine mAb-DNA conjugate, only a decrease of about 9 % was measured showing that, even if fragmentation were indeed induced upon DNA-conjugation, this does not lead to significant loss of the antibody binding affinity.

Figure 5. ELISA analysis of anti-caffeine antibody and DNA conjugate. A) Relative intensity B) Visual result.

Microarray conjugate detection. The last step of this work was to test the immobilization of the produced anti-caffeine mAb-DNA conjugates by DDI. For this purpose both the complementary sequence to that covalently attached to the mAb (ON.B), as well as a noncomplementary probe (ON. C), were immobilized via biotin-streptavidin interaction. The subarrays were blocked with biotin and casein to minimize the unspecific binding of the secondary anti-mouse polyclonal detection antibody, to biotin and to the glass slide, respectively. The results obtained are shown in Figure 6. Two dilutions of the anti-caffeine mAb-DNA conjugate were tested for specific addressing on solid support. The conjugates solution was diluted 1:50 in Figure 6 I) and 1:5 (Figure 6 II).


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When the conjugate solution is diluted 1:5 significant binding background impairs the conjugate detection (Figure 6 II). After a 1:50 dilution of the conjugate, the background binding is decreased and it is possible to distinguish the conjugate being addressed in the rows where the complementary strand is immobilized (Figure 6 III, ON.B row). In addition, no signal is detected in the row where ON.C was immobilized proving that the DDI immobilization of the anticaffeine mAb is highly specific. This experiment clearly demonstrates that immobilization of the obtained conjugates is controlled exclusively by sequence-specific hybridization. Both the non-completion of the DNA conjugation, the random functionalization of the surface antibody and the tendency of this particular antibody to fragment did not impact its successful immobilization via the DNA-DNA specific base pairing. However, room is left for improvement of this detection platform and successful application to other environmentally relevant targets and its compatibility with complex environmental matrices remains to be proven.

Figure 6. DDI of anti-caffeine mAb-DNA immobilized on a streptavidin coated glass slide with microarray detection. Sub-array I. Conjugate dilution 1:50; Sub-array II. Conjugate dilution 1:5; III. Zoom of sub-array I – conjugate addressed using two different concentrations of the capture probe.


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CONCLUSION In this work we illustrate the detailed chemical characterization of antibody conjugates, randomly multi-site functionalized with 12-mer oligonucleotides to be employed for DDI purposes and their subsequent immobilization onto a microarray glass slide. We studied SDSPAGE and MALDI-ToF-MS as complementary strategies to assess the conjugation success and the maximum number of oligonucleotide sequences per antibody molecule, the degree-oflabeling (DOL). Here we have extended our earlier developed conjugation strategy from a humanized diagnostic antibody to a mouse mAb against an exemplary environmental target, the stimulant and important anthropogenic marker caffeine. We could qualitatively assess the integrity of the produced conjugates. Though the employed mouse anti-caffeine mAb was much more prone to fragmentation when conjugated to DNA, this behavior does not seem to greatly affect the binding affinity. As a proof-of-concept for DDI, we were able to immobilize the DNA conjugate on coated glass slides using 12-mer DNA capture probes, which are shorter than the conventionally used ones which are usually 20-mers or even longer ones28,29,33. Short oligonucleotides have the advantage of being easier to synthesize, cheaper and less prone to the formation of complex secondary structures. To the best of our knowledge, this is the first usage of an antibody for the purpose of DDI-based construction of microarrays for environmental analysis. Within this work we foresee the application of such strategies for the future development of high-throughput, well-characterized and established immunoarray platforms for the detection of environmentally relevant contaminants. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected] ORCID Annemieke Madder: 0000-0003-0179-7608 Rudolf J. Schneider: 0000-0003-2228-1248 Ana Margarida Carvalho: 0000-0002-2725-791X


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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/ 2007–2013/ under REA grant agreement No. 316975A. The authors would further like to thank the BOF Special Research Fund from Ghent University, GOA project no. 01G02213. Jos Van den Begin (UGhent) is acknowledged for the synthesis of oligonucleotides employed in this work. Kristin Hoffman (BAM) is acknowledged for ELISA assistance.

ABBREVIATIONS DDI: DNA-directed immobilization; ELISA: Enzyme-linked immunosorbent assay; mAb: monoclonal antibody; MALDI-ToF-MS: Matrix-assisted laser desorption/ionization time-offlight mass spectrometry; ON: oligonucleotide; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate; STV: streptavidin; TCEP: Tris(2-carboxyethyl)phosphine; TMB: 3,3',5,5'Tetramethylbenzidine.

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TABLE OF CONTENTS (TOC)


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An anti-caffeine antibody-oligonucleotide conjugate for DNA-directed

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