Accepted Manuscript Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies Ulrik Boas, Peter Lind, Ulla Riber PII: DOI: Reference:

S0003-2697(14)00288-7 http://dx.doi.org/10.1016/j.ab.2014.07.005 YABIO 11794

To appear in:

Analytical Biochemistry

Received Date: Revised Date: Accepted Date:

6 May 2014 3 July 2014 8 July 2014

Please cite this article as: U. Boas, P. Lind, U. Riber, Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.07.005

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Short title: Conjugation of carbohydrate antigens

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Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies

Ulrik Boas*, Peter Lind, Ulla Riber Section for Immunology and Vaccinology, The National Veterinary Institute, The Technical University of Denmark * Corresponding author: Phone +4535886215; email: [email protected] Abstract A new generic method for the conjugation of Lipopolysaccharide (LPS) derived polysaccharide antigens from Gram negative bacteria has been developed using Salmonella as a model. After removal of Lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene

microbeads modified

with N-alkyl

hydroxylamine

and

N-alkyl O-methyl

hydroxylamine surface groups, by incubation of antigen and beads 16 h at 40 oC without the need of coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies against Salmonella Typhimurium and Salmonella dublin. The presented method was compared to a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide conjugated beads showed preserved bioactivity after one year of use.

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Introduction Gram-negative bacteria, such as Salmonella, Yersinia and E. coli are common pathogens in both humans and domestic animals.1 All these pathogens consist of a spectrum of strains, grouped as serotypes, with divergent infectivity and pathogenicity. Some of the most immunogenic and specific antigens of gram-negative bacteria are capsule and cell wall lipopolysaccharide (LPS) structures – therefore these structures have great potential for producing robust and versatile assays targeting these species, and serotype specific structures.2 However, limitations in assay specificity due to shared carbohydrate antigen structures has been observed and the traditional way of solving this problem has been “blocking” assay formats by competition between hapten-specific monoclonal antibodies and test sera.3 However, another way to increase assay specificity and reduce reactions caused by shared carbohydrate structures would be development of new chemical methods for specific and efficient conjugation of the antigenic determinant polysaccharide structures. 4,5

Chemical coupling of sugars Chemical methodologies to conjugate macromolecular biologically active carbohydrates in a controlled manner without byproducts would provide a valuable and simple tool for the preparation of labeled or immobilized LPS antigens while preserving structural integrity and biological activity. Non-specific conjugation of LPS or polysaccharide (PS) antigens to solid-phases has hitherto been the most employed conjugation strategy. In this strategy, the multiple secondary hydroxyl groups present at the carbohydrate structure are used as tagging points for 1: periodate oxidation to the corresponding “open-chain” poly aldehydes for subsequent reaction with hydrazides (hydrazones) or amines (reductive amination) or 2: by the reaction with cyanuric chloride under alkaline conditions for subsequent reaction with amines on the solid phase.6,7 Both these strategies suffer from being non-specific giving wide spread functionalisation over the entire antigen structure. This 3

non-specificity may significantly compromise antigen activity, and has been addressed as a common flaw of this methodology. 3-Deoxy-D-manno-2-octulosonic acid (KDO) is a sugar moiety that resides in a conserved region of the O-antigen of Gram negative bacterial LPS, the inner core. The inner core is not involved in the recognition of antibodies, and therefore conjugation to KDO should not affect antibody recognition. Therefore, conjugation methods aiming at conjugation to the antigen KDO may show promise also because of the KDO unique structure compared to the other carbohydrate moieties in the antigen structure. Methods for the conjugation to the KDO moiety have been reported earlier.4,5 This method involves the coupling of the polysaccharide antigen to amine surface groups using water soluble carbodiimide and N-hydroxysuccinimide, and is proposed to proceed via the formation of an intermediate KDO carboxylic acid NHS ester which reacts specifically with the amines on the polymer surface. Although the method has shown useful, competing hydrolysis of the antigen KDO NHS ester and/or intramolecular coupling between antigenic functional groups may reduce the efficiency of the coupling of antigen to the surface amines. Alternatively, selective coupling to antigen KDO residues has been obtained by reaction with a hydrazide to form an intermediate acylhydrazone

bond

which

was

subsequently

stabilized

by

reduction

with

sodium

cyanoborohydride.8 However, a coupling method which can be performed under mild conditions without the need of a chemical coupling or reduction agents would be optimal for mild efficient antigen coupling and preservation of antigen structural integrity. In 2006 a new reaction for native peptide ligation was developed by Jeffrey Bode and coworkers. Here, the mixing of two peptide segments, one C-terminally modified with an α-keto acid and the other N-terminally modified with an N-hydroxylamine group, spontaneously forms an amide bond in a polar solvent at 40oC.9-11 As the acyclic ‘keto-form’ of the antigen KDO moiety indeed contains an α-keto acid functionality we reasoned that the Bode native chemical ligation method would be

4

useful for direct conjugation of KDO containing bacterial polysaccharide antigens to N-alkyl hydroxylamines or N-alkyl-O-methyl hydroxylamines (Scheme 1). As the conjugation process is carried out under mild conditions without chemical coupling agents just by gentle heating, the structure of the polysaccharide antigen would not be compromised during the conjugation process, and hence give the optimal conditions for the antibody recognition.

As proof of concept a Salmonella model was used for evaluation of the new chemical coupling of polysaccharide antigens to the surfaces of microbeads to detect antigen specific antibodies. Peculiar monosaccharides, such as abequose (= factor 4 of Salmonella serogroup B) and tyvelose (= factor 9 of Salmonella serogroup D), constitute strongly immunogenic (haptenic) structures in these bacterial lipopolysaccharides. The efficiency of the polysaccharide conjugation method could be evaluated by immunological analysis using flow cytometry, allowing analysis on a “single bead level” to ensure an even antigen coupling to the beads (low coefficient of variation - CV). 12

Materials and Methods Shaking/incubation of beads was carried out on an Eppendorf Thermomixer Compact heater shaker, centrifugation was carried out on a Hettich Mikro 20 centrifuge. Sonication of the beads was performed using a DT 52H, Bandelin Sonorex Digital sonication bath. Washing and staining of the beads were carried out on a Millipore MultiScreen HTS vacuum manifold for 96 well format filterplates (Millipore, MSBVN1210: 1.2µm Hydrophilic, low protein binding Durapore® membrane). Flow cytometric analysis was performed on BD FACS Canto with HTS and Diva 6 software. All chemicals for the chemical coupling procedures were purchased from Sigma-Aldrich 5

and used as received. Amine functionalized microbeads (polystyrene, 3.76µm, 4.57 µeq amino groups/g dry weight beads) were purchased from Spherotech.

13

Modifications of the beads were

carried out in MilliQ water or phosphate buffered saline (PBS). To identify the chemical identity of functional groups on the beads several colorimetric tests were applied. Ninhydrin test (color test for primary amines) was carried out as follows: To a 20 µL of bead suspension one drop of solution A (0.1g ninhydrine in 100mL ethanol) was added followed by one drop of solution B (25 mg Ascorbic acid in 10 mL ethanol) and one drop of a solution C (80g phenol in 100mL ethanol). The mixture was heated for 2 minutes at 80 oC and purple/blue color of the bead suspension indicates the presence of primary amines.14,15 2,4-dinitrophenylhydrazine (DNPH) color test for aldehydes: 2,4 dinitrophenylhydrazine (100mg) was dissolved in concentrated H2SO4 (0.5mL) and this solution was slowly added over one minute to a stirred mixture of water-ethanol 1:10 (7.7 mL). Two drops of this solution were added to 20 µL of bead suspension and the suspension was shaken for 5 minutes. Hereafter the beads were thoroughly washed with MilliQ water (centrifugation and removal of the supernatant, 5 times). Yellow to orange beads indicate the presence of aldehyde groups.16

Delipidation of lipopolysaccharides Delipidation of lipolpolysaccharides from Salmonella Typhimurium and Salmonella dublin and subsequent analysis of the polysaccharide antigen by SDS-PAGE/Silver staining and ELISA was performed according to previously published procedures. 17, 4

Monoclonal antibodies (MAb)

6

For investigation of bioactivity and immunological specificity of beads chemically coupled with polysaccharide antigens from Salmonella Typhimurium (serogroup B) and Salmonella dublin (serogroup D), monoclonal antibodies recognizing factor 4 (sero group B) and factor 9 (serogroup D), respectively, were used. The monoclonal antibodies recognising factor 4 (S. Typhimurium 4,31A15B6) and factor 9 (S. berta 2.36 A14B4C4D2) has been produced and validated at The National Veterinary Institute (DTU-VET).Error!

Bookmark not defined.,Error! Bookmark not defined.

These mAbs were

used to investigate bead coupling capacity and coupling specificity.

Serum samples Furthermore, two bovine serum samples, used as positive controls in serological ELISA Salmonella diagnostics at DTU-VET, were applied

Microbeads with N-alkyl-hydroxylamine surface groups Spherotech amine functionalized microbeads (1mL, 5 % suspension) were centrifuged (3500 rpm, (750g), 1 min, room temperature) and the supernatant was removed by decantation. The beads were suspended in phosphate buffered saline ‘PBS’ (0.6mL, pH 7). Glutaric aldehyde (100 µL, 20% in water) was added followed by sonication bead suspension for 15 minutes to increase the amount of single beads. The mixture was then incubated (shaker, 750 rpm) for 16 h at room temperature. Two small aliquots bead suspension (approx. 25 µL) were taken out, centrifuged (3500 rpm (750g), 1 min, room temperature) and washed three times with PBS, the PBS supernatant was removed and the beads tested by DNPH- and ninhydrin color test. The DNPH test gave yellow beads indicating the presence of aldehyde surface groups on the bead. Here, amine functionalized beads gave colorless beads with DNPH. Ninhydrin test was negative (colorless bead suspension) indicating that 7

primary amines initially present on the beads had been derivatised by glutaraldehyde. With ninhydrin, the amine functionalized beads gave a positive test, visualized by a blue/purple bead suspension.18 After the colorimetric testing, the bead suspension was centrifuged (3500 rpm (750g), 1 minute, room temperature) and the supernatant was removed by decantation. The beads were suspended in PBS (0.6mL, pH 7) and hydroxylamine hydrochloride (24mg) and sodium cyanoborohydride (20mg) were added and the bead suspension was incubated 16 h (shaker 750 rpm) at room temperature. The bead suspension was centrifuged (3500 rpm (750g), 1 minute, room temperature) and the supernatant removed and the beads washed 5 times with MilliQ water. DNPH test showed colorless/off-white beads indicating that aldehyde groups on the beads had been functionalized.

Microbeads with N-alkyl-O-methyl hydroxylamine surface groups Amine functionalized microbeads (0.5mL, 5% suspension) were centrifuged (3500 rpm (750g), 1 min, room temperature) and the supernatant was removed by decantation. The beads were suspended in PBS (0.3mL, pH 7) and glutaric aldehyde (50 µL, 20% in water) was added. The bead suspension was sonicated for 15 minutes and the mixture was incubated (shaker 750 rpm) for 16h at room temperature. Two small aliquots of bead suspension (approx. 25 µL) were taken out, centrifuged and washed three times with PBS, the PBS was removed and the beads tested by DNPH- and ninhydrin color tests. The DNPH test gave yellow beads indicating the presence of aldehyde surface groups on the bead. Ninhydrin test was negative (colorless bead suspension) indicating that primary amines initially present on the beads have been derivatised by glutaraldehyde. The bead suspension was centrifuged (3500 rpm (750g), 1 min, room temperature) and the supernatant was removed by decantation. The beads were suspended in PBS (0.3mL, pH 7).

8

Then methoxyamine hydrochloride (14mg) and sodium cyanoborohydride (10mg) were added and the bead suspension was incubated (shaker, 750 rpm) for 16 h at room temperature. The bead suspension was centrifuged (3500 rpm (750g), 1 minute, room temperature) and the supernatant removed and the beads washed with MilliQ water. The washing procedure was repeated 5 times. DNPH test gave colorless/off-white beads indicating that aldehyde groups on the beads have been functionalized.

Conjugation of polysaccharide antigen to N-alkyl hydroxyl amine beads or N-alkyl-O-methyl hydroxylamine beads (general procedure) N-alkyl hydroxylamine- or N-alkyl-O-methyl hydroxylamine beads were Vortexed for 2 min and 50 µL was transferred to an Eppendorf tube, and polysaccharide antigen in MilliQ water (2mg/mL, 100 µL) was added and the bead suspension was sonicated 15 min to increase the homogenicity of the suspension. Then, the bead suspension was incubated (shaker 750 rpm) for 16 h at 40oC. The beads were transferred to 96 well filter plate placed on a vacuum manifold and washed with 30% aqueous acetic acid (3 x 200 µL), 50% aqueous ethanol (3 x 200 µL) and MilliQ water (3 x 200 µL). Washing procedure was carried out using filterplates and vacuum manifold. The beads were left with washing mixture for 2 minutes before removing the supernatant by vacuum suction. The washing procedures are important to remove small amounts of adsorbed antigen. After the last washing and removal of the supernatant, the microbeads were suspended in MilliQ water (200 µL) and ready for immunological analysis by flow cytometry.

Immunochemical staining

9

Initially suspensions of beads were sonicated for 15 min to optimize single suspension of beads. Staining of beads was performed in 96-well filter plates and washing procedures were performed using a vacuum manifold. Wells in the filter-plate were pre-treated with blocking buffer (PBS, 0.05% Tween 20, 1% BSA) for one min. Beads samples were diluted in blocking buffer and 50µl of bead were added into wells (approx.104-105 beads/well) and incubated at room temperature for 30 min. on a horizontal shaker (Gerhardt, Laboshake). The beads were washed three times (200µl/well) with washing buffer (PBS, 0.05% Tween 20). Dilutions of monoclonal antibodies in blocking buffer (50 µl/well) were added and the plate incubated 30 min at 37°C. The wells were then washed three times with washing buffer and then incubated in dark for 30 min. at approx. 20°C with 50µl/well of reporter antibody (R-phycoerythrin (RPE) conjugated polyclonal rabbit anti mouse Ig (R0439, Dako, Denmark) diluted 1:25 in blocking buffer). Finally three washings were performed and beads were suspended in 250µl sheath fluid (BD FACS FlowTM ). Bead suspensions were transferred into 96-well Falcon plates or tubes for flow cytometric analysis. In experiments using bovine sera as antibody samples, a two-step reporter system was used including 30 min incubation with biotinylated goat-anti-bovine IgG antibody (Rockland) followed by 30 min incubation with RPE-conjugated streptavidin (Invitrogen). Incubations were performed at room temperature at horizontal shaker. Previous titration of the biotinylated antibody and the RPEconjugated streptavidin was performed to find optimal staining conditions, and for the present experiments the biotinylated antibody was used in dilution 1:200 and the RPE-conjugated streptavidin in dilution 1:2000.

Flow cytometry Beads were acquired directly from 96-well plate or from tube. The population of single beads was gated in FSC-A vs. SSC-A with linear scales and minimum 2000 gated events were acquired. The

10

median fluorescence intensity (MFI) of the PE reporter signal was measured in histogram plot showing the gated bead population. To exclude debris thresholds for FSC and SSC were set at 30.000. In most cases the single bead population was around 80-90% of total events, and beadsamples with single beads counting less than 70% were not taken into account. Results and discussion Modification of amine microbeads: Polystyrene microbeads (3.76 µm) with surface amine groups (4.57 µeq amines/g dry weight) were applied and several approaches were investigated to transform the amine surface groups to N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups respectively. Initially, the amine groups on the beads were alkylated with 1,3dibromopropane in ethanol followed by reaction of the ‘bromo’ functionalised bead surface with hydroxylamine or methoxyamine hydrochlorides in water. This method gave promising results upon subsequent conjugation to the polysaccharide antigen with high sensitivity and specificity in antibody recognition in the flow cytometry assay. However, the method was not generally applicable for the surface modification of fluorescent microbeads as these broke down in organic solvents. Therefore, we developed a more generic method for the modification of the beads, which could be carried out in aqueous solution. This method was based on initial reaction of the amine beads with aqueous glutaric aldehyde. As the concentration of functional groups on each bead is very low (≤4.6 µmol/g dry beads), the functionalization of the beads surface groups was difficult to monitor by spectroscopic methods such as infrared spectroscopy (IR). When analysing the beads by IR, we observed that the spectrum of the functionalized bead was identical to spectrum of the initial amine functionalized bead before functionalization. Therefore, qualitative colorimetric tests for functional groups were applied instead and proved useful. The presence of aldehyde groups on the bead surface could be monitored colorimetrically by the use of 2,4-dinitrophenylhydrazine (DNPH) test to give deeply yellow coloured beads. The aldehyde modified beads were then subjected to

11

sodium borohydride mediated reductive ‘hydroxylamination’ with hydroxyl amine and methoxyamine, respectively. After the reductive hydroxylamination step the DNPH test gave colorless beads, indicating that aldehyde groups on the bead surface had been transformed.

The optimal conditions for the formation of the N-alkyl O-methyl hydroxylamine linker beads were investigated by flow cytometry. Here, we assumed that an efficient synthesis of the reactive linker on the bead should subsequently give a more effective conjugation of antigen and thereby an optimal reporter signal after incubation with antigen specific monoclonal antibodies.19 We found that beads which had been subjected to only 3 hours of glutaric aldehyde treatment at room temperature gave considerably lower reporter signal (i.e. lower conjugation of antigen), compared to beads which were treated with glutaric aldehyde for 3 hours at 50oC. However, the latter method induced increased background (auto-fluorescence), whereas beads which were treated 16 h with glutaric aldehyde at room temperature gave the best performance.

Effect of time, temperature and coupling volume on capacity of chemical coupling: To establish the optimal condition for the conjugation of antigen to the modified micro-beads, the conjugation processes were carried out at 20°C, 40°C and 60°C with a fixed reaction time of 16 h. (Figure 1). It was found that optimal efficiency of antigen conjugation was achieved at 40°C, whereas lower coupling efficiencies were obtained at 20°C and 60°C respectively. By raising the temperature to 60°C increased autofluorescence was also observed.

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Optimal antigen conjugation time was found to be around 16h, whereas 4 h coupling time showed lower reporter signal and a 2-day coupling period generally increased the autofluorescence of the beads (data not shown).20 The effect of chemical coupling on the beads may vary dependent of reaction volume, as the reaction is heterogeneous, the bead surface groups may become more accessible with a lower bead concentration due to reduced aggregation/clotting of the beads. Therefore, we varied the reaction volume to find the volume which was optimal for maximum exposure of the bead surface functionalities together with minimum bead aggregation (Figure 2).

Here we found that a larger reaction volume (lower concentration of the antigen) resulted in a lower sensitivity of the beads in the recognition of antibodies. This indicates that, albeit the bead suspension was more dilute to prevent aggregation of the beads, a lower concentration of antigen also gave a lower ‘antigen-loading’ on the beads. To determine the efficacy of the new conjugation method, the level of unspecific antigen adhesion to the beads was investigated. Unmodified amine beads and N-alkyl hydroxyl amine/N-alkyl-Omethyl hydroxyl amine beads (‘linker beads’) were incubated with similar amounts of polysaccharide antigen (S. Typhimurium) at 40oC for 16 h and the beads were subjected to similar washing steps. After incubation with specific MAb and reporter antibody the linker beads conjugated with S. Typhimurium antigen showed much higher reporter signal compared to the amine beads subjected to unspecific adsorption of antigen. This is an indication that the main immune reactivity was obtained by conjugation of antigen, although a minor degree of unspecific 13

antigen adsorption on the unmodified amine beads was also observed (Figure 3). By testing different ‘post incubation’ washing procedures we found that washing of the beads with 50% ethanol in MilliQ water was crucial to remove unspecific adsorbed antigen (data not shown). Washing the beads with acetic acid had less effect but was introduced to potentially break down ionic interactions between charged functional groups (e.g. phosphates, amines and carboxylic acids) at the antigen and charged bead surface groups.

The exact mode of binding between the antigen and the surface groups was difficult to determine by spectroscopic methods, due to the low loading of antigen on the bead surface. Also here we carried out infrared spectroscopy using attenuated technique of reflection (IR-ATR) on the antigen beads and found, as with the N-alkyl hydroxylamine/N-alkyl-O-methyl hydroxylamine modified beads, that the IR spectra of the antigen beads were identical to the initial amine functionalized bead exclusively showing the spectrum of polystyrene. On-resin spectroscopic analysis such as IR-ATR and magic angle spinning nuclear magnetic resonance (MAS-NMR) can be carried out with success when performing solid-phase synthesis on resins with functional group loadings ranging from approximately 0.3 mmol/g. The functional group loading present at the microbeads applied in this study is, however, magnitudes lower in comparison.21 Therefore, we applied immunochemical staining combined with flow cytometry to give an indication of what chemical functionalities of the antigen could be involved in the conjugation to the bead surface groups. As the reaction between antigen and N-alkylhydroxyl amine and N-alkyl-O-methyl hydroxylamine surface groups is proposed to happen via a KDO α-keto acid on the antigen, blocking the surface groups by

14

incubation with an excess of an α-keto acid “dummy” was investigated (phenyl pyruvic acid, figure 4).

After incubation of the N-alkyl-O-methyl hydroxylamine beads with phenyl pyruvic acid we indeed observed a significantly lower efficiency in the subsequent binding of polysaccharide antigen to the surface, also after washing the beads with ethanol and acetic acid (Figure 5). However, with the Nalkyl hydroxylamine beads no significant reduction of the subsequent binding of the polysaccharide antigen was observed. Although, the experiment give some indication that the KDO α-keto acid may be involved in the binding of the antigen to the beads surface groups, it is not fully conclusive, and factors such as hydrophobicity of the bead surface groups and phenylpyruvic acid may play a role as well.

We compared the present method with a previously published procedure for the conjugation of polysaccharide antigens to amino groups via the KDO residue by water soluble carbodiimide/Nhydroxysuccinimide as coupling agents (scheme 1).4 We found that the new method gave beads with a higher sensitivity, i.e. were able to detect lower Typhimurium MAb concentrations (Figure 6). In the conjugation of S. dublin antigen we found that the new method gave more uniformly coupled beads (70-80% single beads) compared to S. dublin beads prepared by the previously reported method which induced aggregation yielding less than 50% single beads.18 Here, it may be speculated that the latter method being dependent on coupling agent induce some extent of antigen and bead cross-linking which is not as abundant when applying the new method. 15

Immunochemical test signals were increasing with increasing amount antigen coupled to beads (for antigen concentrations below saturation). Generally low cv’s in reporter signal for bead populations was found, indicating an equal distribution of antigen on the single beads, however, for bead populations with low “reporter signal” due to reduced amount of bound antigen or low level of sample antibodies the cv’s were increased compared to bead populations with high reporter signals. To evaluate the utility of the new conjugation method in the serological analysis of serum samples, beads coupled with S. Typhimurium and S. dublin antigen and beads with no antigen were incubated with serum samples known to be positive in ELISA tests (Figure 7). The positive serum samples was diluted in negative serum to give a specific ELISA reading, as these sample are used as positive and negative controls in diagnostic tests for Salmonella antibodies. Hence, a decreased reporter signal with increasing serum sample dilution was observed. Compared to the negative serum sample beads coupled with S. Typhimurium showed significantly increased reporter signal only for the S. Typhimurium serum sample, whereas the S. dublin serum sample was at a similar level as the negative serum sample. For beads coupled with S. dublin antigen, the S. dublin serum sample showed significantly increased reporter signals compared to the negative serum sample and the S. Typhimurium positive serum sample. However, for S. Typhimurium serum sample slightly increased reporter signals were observed when tested with beads not conjugated with antigen (Figure 8). .

16

Upon titration of serum antibodies, the S. Typhimurium, S. Dublin and beads without antigen gave specific reaction even at high Typhimurium serum antibody concentrations. Here, the Typhimurium beads gave a positive signal whereas Dublin beads and beads without antigen gave low signal. However, upon testing the beads towards a dublin serum, some cross reactivity was observed at the highest antibody concentrations (Figure 8). The cross reactivity may be related to the factor 12 saccharide residues [2β-D-Man-1→4-α-L-Rha4-1→3-β-D-Gal (4←1-α-Glc)-1] common for both the S. dublin and S. Typhimurium O-antigens.22, 23

In summary, a new method for the mild conjugation of Salmonella LPS derived polysaccharide antigens to N-alkylhydroxyl amine and N-alkyl-O-methyl hydroxylamine surface groups on microbeads has been developed and evaluated. The conjugation can be carried out without the need of coupling agents. In a comparison with a previously published method the presented method led to more efficient coupling of the Salmonella antigen to microbeads, where the known method based on coupling agents resulted in beads with lower sensitivity towards monoclonal antibodies in model samples than the present method. In the coupling of Salmonella dublin the method based on coupling agents showed bead aggregation and gave a much poorer coupling efficiency on single beads compared to the new method. The new method led to low cross reactivity in the exposure of Typhimurium beads to antibodies against S. dublin (S. Berta) and vice versa. Also, when exposed to serum samples the antigen beads prepared by the present method showed good sensitivity towards antibodies in the samples. Stability studies indicate that antigen beads that have been stored for 1 year still have sensitivity which is of the same magnitude (±20%) as freshly prepared antigen beads. The present method is a mild and stable conjugation method which will be widely applicable in the

17

coupling of KDO containing Gram negative antigenic polysaccharides. Furthermore, our results indicate that the beads coupled with antigen by the present method can be used for diagnostic testing of serum samples opening for possible use in multiplex analysis using fluorescent beads. However, optimization regarding sample dilution and reporter staining procedures are required to obtain optimal P/N results for samples. Preliminary conjugation using fluorescent beads has shown the mild conjugation method not to affect the bead fluorescence, which stayed at similar level for conjugated beads and control beads (data not shown) and further studies to implement the method in multiplex analysis of Gram negative antigens will be pursued in the future. Acknowledgements Lars Ole Andresen is gratefully thanked for carrying out fermentation of salmonella bacteria. Jeanne Toft Jakobsen is thanked for excellent laboratory assistance on bead analysis by flow cytometry and Abdellatif El Ghazi is thanked for excellent assistance in the purification and delipidation of LPS from Salmonella. Ulrik Boas and Ulla Riber is grateful to The Danish Research Council for Technology and Production (FTP) for financial support (grant 09-064237)

References [1] E.T. Rietschel, T. Kirikae, F. U. Schade, U. Mamat, G. Schmidt, H. Loppnow, A. J. Ulmer, U. Zähringer, U. Seydel, F. Di Padova, M. Schreier, H. Brade, Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8 (1994) 217-225 [2] I. M. M. Helander, V. Kitunen, Cleavage of the O-antigen 4,5,12 of Salmonella Typhmurium by hydrofluoric acid, FEBS Lett. 250 (1989) 565-569

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[3] J. Hoorfar, N.C. Feld, A.L. Schirmer, V. Bitsch, P. Lind, Serodiagnosis of Salmonella Dublin infection in Danish dairy herds using O-antigen based enzyme-linked immunosorbent assay. Can. J. Vet. Res. 58 (1994) 268-274. [4] E.S. Jauho, U. Boas, C. Wiuff, K. Wredstrøm, B. Pedersen, L. O. Andresen, P. M. H. Heegaard, M. H. Jakobsen, New technology for regiospecific coupling of polysaccharide antigens in ELISA for serological detection. J. Immunol. Meth. 242 (2000) 133-143 [5] C. Wiuff; E. S. Jauho, H. Stryhn, L. O. Andresen, K. Thaulov, U. Boas, M. H. Jakobsen, P. M. H. Heegaard, Evaluation of a novel enzyme-linked immunosorbent assay for detection of antibodies against Salmonella, employing a stable coating of Lipopolysaccharide-derived antigens covalently attached to polystyrene wells, J. Vet. Diagn. Invest. 12 (2000) 130-135 [6] R. E. Biagini, S. A. Schlottmann, D. L. Sammons, J. P. Smith, J. C. Snawder, C. A. F. Striley, B. A. MacKenzie, D. N. Weissman, Method for simultaneous measurement of antibodies to 23 pneumococcal capsular polysaccharides, Clin. Diagn. Lab. Immunol. 10 (2003), 744-750 [7] G. Laher, P. Balmer, S. J. Gray, M. Dawson, E. B. Kaczmarski, R. Borrow, Development and evaluation of a rapid multianalyte particle-based flow cytometric assay for the quantification of meningococcal serogroup B-specific IgM antibodies in sera for nonculture case confirmation, FEMS Immunol. Med. Microbiol. 48, (2006) 34-43 [8] F. Micoli, S. Rondini, M. Gavini, L. Lanzilao, D. Medaglini, A. Saul, L. B. Martin, O:2CRM197 conjugates against Salmonella Paratyphi A, PloS ONE 7 (2012) e47039 [9] J. W. Bode, R. M. Fox, K. D. Baucom, Decarboxylative condensations of alpha-ketoacids with N-alkylhydroxylamines: A new amide ligation reaction, Angew. Chem. Int. Ed. Engl. 45 (2006) 1248-52

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[10] J. W. Bode, Emerging methods for the synthesis of amide and peptide bonds, Curr. Opinion Drug Discov. Dev. 9 (2006) 765-775 [11] V. Pattabiraman, J. W. Bode, Rethinking amide bond synthesis, Nature 480 (2011) 471-479 [12] Bead population with low spread in signal due to similar amount of antigen coupled to each bead result in a low CV [13] Information can be found on Spherotech homepage: www.spherotech.com [14] E. Kaiser, R.L. Colescott, C. D. Bossinger, P.I. Cook, Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides, Anal. Biochem. 34 (1970) 595-598 [15] S. Yokoyama, J.-I. Hiramatsu, A modified ninhydrin reagent using ascorbic acid instead of potassium cyanide, J. Biosci. Bioeng. 95 (2003) 204-205 [16] S. K. Shannon, G. Barany, Colorimetric monitoring of solid-phase aldehydes using 2,4dinitropenylhydrazine, J. Comb. Chem. 6 (2004) 165-170 [17] F. M. Unger, The Chemistry and biological significance of 3-deoxy-D-manno-2-octulosonic acid (KDO), Adv. Carb. Chem. Biochem. 38 (1981) 323-388 [18] See supporting information [19] Fixed conjugation conditions: Antigen conjugation for 16 hours at 40oC [20] In these experiments, the amount of antigen (100 µg) and coupling volume (75 µl) were fixed. Immunochemical testing, paired experiment, was performed using antigen specific mAb (Typh) in two concentrations as well as unspecific mAb (Berta). [21] For the use of MAS-NMR in solid-phase synthesis see e.g: R. Hany, D. Rentsch, B. Dhanapal, D. Obrecht, Quantitative determination of resin loading in solid-phase organic synthesis using

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C MAS NMR, J. Comb. Chem. 3 (2001) 85-89 and L. R. Carvalo, M. C. Corvo, R. Enugala,

M. M. B. Marques, E. J. Cabrita, Application of HR-MAS NMR in the solid-phase synthesis of a glycopeptide using Sieber amide resin, Magn. Reson. Chem. 48 (2010) 323-330 [22] F. G. van Zijderveld, A. M. van Zijderveld- van Bemmel, J. Anakotta, Comparison of four different enzyme-linked immunosorbent assays for serological diangosis of salmonella enterididis infections in experimentally infected chickens, J. Clin. Microbiol. 30 (1992) 25602566 [23] P. A. D. Grimont, F. Grimont, P. Bouvet. Taxonomy of genus salmonella. Chapter 1 in Salmonella in domestic animals (eds: C. Wray, A. Wray.) CABI Publishing 2000

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U. Boas, P. Lind, U. Riber. Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies

Figure legends Scheme 1. Proposed scheme for the modification and conjugation of polysaccharide antigen via the KDO residue to micro beads with N-alkyl hydroxyl amine and N-alkyl-O-methyl hydroxyl amine surface groups, together with the previously published water-soluble carbodiimide (WSC)/ N-hydroxysuccinimid (NHS) mediated coupling method for Salmonella polysaccharide antigens.

Scheme 2. Proposed chemical scheme for the transformation of bead surface amine groups to N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxyl amine surface groups, respectively

Figure 1. Coupling capacity as a function of temperature (S. Typhimurium beads). A: MAb Typhimurium 1µ g/mL. B: MAb Typhimurium 0.2 µ g/mL. C: MAb Berta 1µg/mL. D: No MAb. Dotted bar: 20oC. Black bar: 40oC. Striped bar: 60oC

Figure 2. Bead coupling efficiency as function of reaction volume. Beads were coupled with 2-fold dilutions (2mg/ml – 0.25mg/ml) of Salmonella Typhimurium antigen (PS) as standard procedure i.e. 25μl linker beads and 50μl antigen suspension in total of volume 75μl or with addition of MilliQ water ad 225μl. Control beads were prepared without addition of antigen. The antigen capacity of the beads was tested by flow cytometry after incubation with antigen-specific monoclonal antibody (Mab) in two concentrations,

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1μg/ml (black squares) and 0.2μg/ml grey squares) or without Mab (white squares) followed by incubation with RPE-conjugated reporter antibody as described in Materials and Methods. Antibody staining was performed in triplicate in paired experiments. The data is shown as mean+/- SD.

Figure 3. Conjugation of S. Typhimurium antigen to functionalized beads by new chemical coupling compared to passive adsorption to amino beads. Antigen (12.5μg - 100μg) was incubated with beads in optimal coupling volume (75μl). Incubations were performed at 40°C. Reporter signals are shown for, grey bars: Antigen conjugation to functionalized beads and white bars: Passive antigen adsorption to amino beads both with antigen specific mAb (1μg/ml). Striped bars: Incubation with unspecific mAb (1μg/ml). Line indicates background level (mean + 2SD) of beads incubated without Mab. Bead antigen was only detected by the specific monoclonal antibody (S. Typhimurium; factor 4) whereas using unspecific monoclonal antibody (S. Berta; factor 9) showed reporter signals at background level.

Figure 4. Structure of phenylpyruvic acid

Figure 5. Effect of preincubation with phenylpyruvic acid prior to conjugation of S. Typhimurium antigen to N-alkyl-O-methyl hydroxylamine modified beads. A: Conjugation of antigen without preincubation with phenylpyruvic acid. B: Conjugation of antigen after preincubation with phenylpyruvic acid. C: Preincubation with phenylpyruvic acid, but no antigen added. Dotted line: Background

Figure 6. Sensitivity of antigen coupled beads detecting antibodies in samples containing different levels of specific or unspecific monoclonal antibody. Beads were coupled with PS from S. Typhimurium or S. dublin as described in Material and Methods. Grey circles: S. Typhimurium beads incubated with dilution of

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specific antibody, i.e. mAb against factor 4 and S. dublin beads incubated with mAb against factor 9. Open circles: Amine beads coupled with same amount of antigen using water soluble carbodiimide were tested in paired experiment and reporter signals shown. Dublin antigen beads prepared by water soluble carbodiimide gave too low level of single beads (around 50%) and were not taken into account. Triangles: Incubation of beads with the unspecific monoclonal antibody, factor 9 for S. Typhimurium beads and factor 4 for S. dublin beads, giving signals at background levels. The figure shows a linear signal response to 2-fold dilutions of specific monoclonal antibody with both axes in log scale. Line indicates background level (mean + 2 SD) measured in response to beads incubated without monoclonal antibody. Incubations were performed in duplicates and mean +/- SD are shown.

Figure 7. Test of the antigen coupled beads for detection of Salmonella antibodies in serum samples. Serum samples were diluted 1:25 – 1:1600 in blocking buffer and tested as described in materials and methods. X-axes are shown in log-scale and Y-axes in linear scale. Line indicated the background level for beads incubated without serum samples. Immunochemical staining was performed in duplicate and shown as mean +/- SD. Open square: S. Typhimurium serum. Grey square: S. Dublin serum. Black triangle: Negative serum

Figure 8. Test of the antigen coupled beads for detection of Salmonella antibodies in serum samples.. Serum samples were diluted in Salmonella negative serum followed by analysis in a 1:100 dilution in blocking buffer and tested as described in Materials and Methods,. The mean reporter signal for the negative serum (used for dilution of positive sera) is indicated by the lines, beads without antigen (......) and beads with S. Typhimurium or S. dublin antigen (-----). The figure show reporter signals from testing dilutions of serum antibodies on beads with or without antigen coupling (both axes in log scale). Black triangles: Beads without antigen. Open squares: S. Typhimurium beads. Grey squares: S. dublin beads.

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Scheme 1

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Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies.

A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed us...
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