a repertoire of high affinity monoclonal antibodies specific to s. typhi

8/15/2019 A Repertoire of High Affinity Monoclonal Antibodies Specific to S. Typhi

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A repertoire of high-afnity monoclonal antibodies specicto S. typhi : as potential candidate for improved typhoid diagnostic

Chandresh Sharma 1 • Anurag Sankhyan 1 • Tarang Sharma 1 •

Naeem Khan2

• Susmita Chaudhuri1

• Niraj Kumar1

•

Shinjini Bhatnagar 3 • Navin Khanna 4 • Ashutosh Tiwari 1

Springer Science+Business Media New York 2015

Abstract Typhoid fever is a signicant global healthproblem with highest burden on the developing world. Theseverity of typhoid is often underestimated, and currentlyavailable serological diagnostic assays are inadequate dueto lack in requisite sensitivity and specicity. This under-lines an absolute need to develop a reliable and accuratediagnostics that would benet long-term disease controland treatment and to understand the real disease burden.Here, we have utilized agellin protein of S. typhi that issurface accessible, abundantly expressed, and highly im-munogenic, for developing immunodiagnostic tests. Flag-ellin monomers are composed of conserved amino-terminaland carboxy-terminal, and serovar-specic middle region.We have generated a panel of murine monoclonal anti-bodies (mAbs) against the middle region of agellin, pu-ried from large culture of S. typhi to ensure its nativeconformation. These mAbs showed unique specicity andvery high afnity toward S. typhi agellin without showingany cross-reactivity with other serovars. Genetic analysisof mAbs also revealed high frequency of somatic mutationdue to antigenic selection process across variable region toachieve high binding afnity. These antibodies also

displayed stable binding in stringent reaction conditions forantigen–antibody interactions, like DMSO, urea, KSCN,guanidinium HCl, and extremes of pH. One of the mAbspotentially reversed the TLR5-mediated immune response,in vitro by inhibiting TLR5–agellin interaction. In ourstudy, binding of these mAbs to agellin, with high af-nity, present on bacterial surface, as well as in solubleform, validates their potential use in developing improveddiagnostics with signicantly higher sensitivity andspecicity.

Keywords Flagellin S. typhi Typhoid fever

Monoclonal antibodies TLR5 Typhoid diagnostics

Introduction

Typhoid, an enteric fever of humans, is a systemic life-threatening infection caused by the bacteria Salmonellaenterica serovar Typhi ( S. typhi ) [1]. Occurrence is morecommon in the lesser-developed regions of the world,where overcrowding and inadequate sanitation are preva-lent [ 2]. According to the best global estimates, the annualincidence of typhoid fever is approximately 27 million

cases, with 216,000 deaths [ 3, 4]. The major proportion of this burden is contributed by the citizens of low-incomecountries, particularly those in Southeast Asia, Africa, andLatin America. India along with Pakistan and Bangladeshaccounts for about 85 % of the world’s cases with a highestincidence in preschool children [ 5–8]. Contaminated foodand water are responsible for the disease which is main-tained in human population through carriage [ 9, 10]. Esti-mates vary, but potentially 1–5 % of patients with acuteinfection have been reported to become chronic carriers, as

& Ashutosh [email protected]

1 Centre for Bio-design and Diagnostics, Translational HealthScience and Technology Institute, NCR Biotech ScienceCluster, 3rd Milestone, Faridabad-Gurgaon Expressway,Faridabad 121001, Haryana, India

2 National Institute of Immunology, New Delhi, India3 Pediatric Biology Center, Translational Health Science and

Technology Institute, Faridabad, Haryana, India4 International Centre for Genetic Engineering and

Biotechnology, New Delhi, India

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Immunol ResDOI 10.1007/s12026-015-8663-z

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Fig. 1 Construction, purication, and characterization of native andrecombinant agellin protein. a Illustration of aligned amino acidsequence of agellin ( S. typhi , S. typhimurium , and S. paratyphi ),showing conserved and hyper-variable middle region (shaded ingrey ). b Illustration of cloning of iC gene encoding agellin (fulllength) and deletant constructs ( middle region ) in E. coli expressionvector pPROEXHTb, for expression of recombinant agellin. cProtein expressed in soluble (cytosolic) fraction was IMAC-puriedand analyzed on 15 % Tris–glycine, SDS-PAGE followed by staining

with Coomassie Brilliant Blue, from different sources ( S. typhi and S.typhimurium ): molecular mass marker ( lane 1 ), * 52-kDa recombi-nant full-length His-tagged agellin ( lane 2 ), and * 25-kDa recom-binant middle-region His-tagged agellin ( lane 3 ). d Western blotanalysis using anti-His monoclonal antibody (Cell Signaling). eIsolation of native agellin from S. typhi and S. typhimurium ;molecular mass marker ( lane 1 ) and * 52-kDa native tagged agellinprotein ( lane 2 ) were analyzed on 15 % Tris–glycine, SDS-PAGEfollowed by staining with Coomassie Brilliant Blue

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cloned into respective sites in the MCS of pPROEX TM

HTb vector (Invitrogen, USA) to construct plasmidpPROEXHTb- iC (full length; 1-512 AA) and pPROEXHTb- iC (middle region; 144-316 AA), for the expressionof S. typhi or S. typhimurium recombinant agellin. Plas-mid constructs made were sequenced, which showed100 % sequence homology with their respective iC gene.

Purication of recombinant agellin protein

Protein was expressed by subsequently transformingplasmid constructs into chemically competent E. coli Rosetta-gami

TM (DE3) cells (Novagen–Merck, Darmstadt,Germany). Briey, overnight culture was inoculated in 1 Lof fresh 2X TY media (Difco, USA) (1:100) and inducedby 0.5 mM IPTG (Amresco LLC, OH, USA) overnight at37 C. Cells were collected and centrifuged, and the pelletobtained was resuspended in buffer [phosphate-bufferedsaline (pH 7.4), 1 mM PMSF]. Cells were lysed bysonication. Supernatant obtained post-centrifugation wasused for purication of recombinant agellin protein byimmobilized metal ion afnity chromatography (IMAC)using HisTrap FF column (GE Healthcare Bucking-hamshire, UK), as per manufacturer’s protocol. Protein waseluted using buffer [phosphate-buffered saline (pH 7.4),250 mM imidazole]. Puried protein was dialyzed against

phosphate-buffered saline (pH 7.4), for the removal of imidazole and then concentrated. Protein was quantitatedby bicinchoninic acid assay (Thermo Scientic Pierce,Rockford, IL, USA) and analyzed using 15 % Tris-glycine, SDS-PAGE, and conrmed by Western blotting,using anti-His monoclonal antibody (Cell Signaling Tech-nology, Inc., MA, USA).

Isolation of native agellin

Bacteria grown overnight in 20 ml of Luria–Bertani (LB)media (Difco, USA) was inoculated in 1 L of fresh LBmedia (1:1000) and incubated overnight at 37 C. Cellswere harvested by centrifugation at 5000 9 g for 30 min,washed thrice with PBS, and nally resuspended in PBS.Native agellin from the S. typhi was isolated using earlierpublished method with slight modications [ 33]. Briey,cells were subjected to mechanical tearing and supernatantwas collected by centrifugation. Two steps of ultracentrifu-gation of supernatant at 100,000 9 g followed by heat inac-tivation for 1 h at 70 C and nal ultracentrifugation yieldedpure native agellin. Western blotting, using anti-agellinmonoclonal antibody, conrmed isolation of native agellin.

Generation, purication, and characterizationof anti-agellin mAbs

All the animal experiments in this study were carried out atInternational Centre for Genetic Engineering andBiotechnology, New Delhi, India, and in accordance withthe guidelines of Institutional animal ethics committee. Agroup of female Balb/c mice (age; 6–8 weeks) were im-munized subcutaneously (s.c.), with puried native ag-ellin (20 l g in 100 l l PBS per animal), from S. typhi alongwith Freund’s Complete Adjuvant (CFA 1:1). Mice wereboosted four times with antigen (10 l g in 100 l l PBS peranimal), along with Freund’s incomplete Adjuvant (IFA1:1). Mice were bled 3 days after the last booster, and themouse with the highest titer of serum antibodies to nativeagellin antigen was given nal booster injections, in-traperitoneally (i.p.), 4 days before aseptically removingthe spleen. Splenocytes were utilized in hybridoma gen-eration, for preparation of monoclonal antibody using theClonacell-HY system (Stemcell Technologies, USA), ac-cording to manufacturer’s instructions. Once established,the clones were expanded in tissue culture asks and storedin liquid nitrogen for future use. Hybridomas producinganti-agellin antibodies were rescreened and puried twotimes by limiting dilution. The hybridoma culture soup wasclaried, and anti-agellin mAbs (IgG) were puried byprotein G afnity chromatography. Puried mAbs weredialyzed against phosphate-buffered saline (pH 7.4) andconcentrated. Protein was quantitated by determining the

Fig. 2 Purication and characterization of S. typhi anti-agellinmonoclonal antibodies; Purication of anti-agellin mAbs (IgG) wasperformed by protein G afnity chromatography a Tris–glycine, SDS-PAGE (15 %) followed by staining with Coomassie Brilliant Bluerevealed the corresponding band of heavy and light chain at * 50 and* 25 kDa, respectively; molecular mass marker ( lane 1 ), mAb cloneP4D9 ( lane 2 ), mAb clone P10F11 ( lane 3 ), mAb clone P8F3 ( lane 3 ),and mAb clone P8F10 ( lane 4 ). b Antigen-binding analysis and titers(IgG levels) for mouse mAbs were determined by ELISA. Threemonoclonals P4D9, P10F11, and P8F3 showed very good andcomparable titers, while P8F10 had least titers. Error bars representthe standard error calculated from triplicate measurements. c Screen-ing and binding analysis of mAbs to the pure native, recombinant, andrecombinant middle-region agellin as shown by ELISA. Data areexpressed as mean ± SE calculated from triplicate measurements.d Immunoblotting was performed using pure native ( lane N ),recombinant ( lane R ), and recombinant middle-region ( lane M ) ag-ellin from S. typhi or S. typhimurium for the demonstration of specicity in detection of agellin (in denaturing condition) by thedifferent monoclonal antibodies. Upper panel shows the immunoblot-ting analysis with native isolated agellin; in the lower panel, purenative, recombinant, and recombinant middle-region agellin wereused, from both bacterial serovars. e Binding analysis of mAbs to

agellin attached on bacterial surface performed by ow cytometry.Negative control shows unstained cells (having PBS only). Data areexpressed as histogram and also with bar diagram (f ), a shift in the peak of histogram represents the binding of mAbs to the agellinattached on bacterial surface. All the four mouse monoclonal antibodydid not cross-react with E. coli (non-related antigen control) agellin.Data are a representation of one of the best results, obtained fromthree separate experiments

b

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Fig. 2 continued

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OD value at 280 nm, using Nanodrop 2000c spectropho-tometer (Thermo scientic, DE, USA) and analyzed using15 % Tris–glycine, SDS-PAGE. Screening and bindinganalysis of mAbs to the pure native, recombinant, and re-combinant middle-region agellin was performed byELISA. NUNC Maxisorp ELISA plate was coated withantigen (1 l g ml

- 1 ) in 100 l l bicarbonate buffer (pH 9.5)and incubated overnight at 4 C. Plates were blocked with5 % non-fat milk in PBS for 2 h at 37 C. Monoclonalantibodies (100 l l per well), (a) hybridoma supernatantand (b) puried mAbs, in different dilutions, were added inantigen-coated plates and incubated for 1 h at 37 C.Levels of bound antibodies were detected, by conjugatedanti-mouse IgG-HRPO antibody (1:2500; Cell Signaling)using TMB (3,3 0,5,5 0-tetramethylbenzidine) substrate. Thereaction was stopped after 10 min by 2N H 2 SO 4 (50 l l perwell), and optical density (OD) was measured at 450 nm.

Immunoblotting with anti-agellin mAbs

Specicity in detection of S. typhi agellin wasperformedbyimmunoblotting. Pure native, recombinant, and recombinantmiddle-region agellins from S. typhi or S. typhimuriumwere resolved on a 15 % reducing Tris–glycine, SDS-PAGEand transferred to nitrocellulose membrane (GE Healthcare,Buckinghamshire,UK). After blocking with PBS containing5 % fat-free milk powder for 2 h, different sources of ag-ellin were detected, using anti-agellin mAbs (1/5000 dilu-tion), and secondary antibody: anti-mouse HRP conjugate(1/10,000 dilution, Cell Signaling Technology, Inc., MA,USA), using Amersham ECL Prime Western Blotting de-tection reagent (GE Healthcare, Buckinghamshire, UK).

Binding of mAbs to the agellin attachedon bacterial surface

All enterobacterial strains were grown overnight in 20 ml of LB media. Bacterial cells (5 9 10 7 ) were incubated on icewith different anti-agellin mAbs (1:500 dilution), for 1 h.Cells were washed twice, stained, with uorochrome-con- jugated monoclonal goat anti-mouse IgG antibody (1:200;dilution): Alexa Fluor 488 goat anti-mouse IgG (H ? L)(Thermo Fisher Scientic Inc. MA, USA), for 1 h, and xedwith 2 % paraformaldehyde. Bound anti-agellin mAbs weredetected on FACSVerse (BD Biosciences) ow cytometer,and the data were analyzed using FlowJo software (v.10.0.5).E. coli (strain XL1-blue) was used as negative control.

Antigen-binding strength and stability of S. typhianti-agellin mAb

The antigen-binding strength of anti-agellin mAb wasassayed qualitatively by ELISA in the presence of various

destabilizing agents such as urea (0–6 M), DMSO(0–10 %), NaCl (0–4 M), potassium thiocyanate (0–3 M),over a wide pH range (2.5–9). All buffers used werephosphate-buffered saline (PBS) based. The destabilizingagents and their ranges were chosen as in Reference [ 34].

Afnity measurement of S. typhi anti-agellin mAb

Antigen–antibody interaction kinetics analysis was per-formed using SPR analysis on ProteOn XPR36 System(Bio-Rad, California, USA). ProteOn GLM sensor chipwas conditioned with 30 l l of acetate buffer (pH 4.5) for60 s, in order to get stable baseline in all the six channels.ProteOn GLM sensor chip was chemically activated by theinjection of 30 l l an equivolume mixture (1:1) of 400 mM1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)and 100 mM N-hydroxysulfosuccinimide (NHS). Subse-quently, 30 l l of different S. typhi anti-agellin mAbs(5 l g ml

- 1 diluted in acetate buffer; pH 4.5) were injectedin four channels for 200 s to get an effective immobiliza-tion of anti-agellin mAbs, on the activated ProteOn GLMsensor chip surface. Following antibody immobilization,the remaining active sites were then blocked with 30 l l of 1 M ethanolamine. Afterwards, 10 mM HCl was injectedto achieve regeneration of immobilized sensor surface. Fornegative control measurements, the modied gold surfacewas activated with EDC/NHS and then quenched withethanolamine in channel 5 as mentioned above and wasused as blank control surface. In order to deduce kineticparameters, native agellin of S. typhi , in ve differentconcentrations (100, 33.33, 11.11, 3.70, and 1.23 nM), wasowed in PBS (pH 7.4), over the antibody-immobilizedsensor chip and interacted with different concentrations of antigen. The interaction of antibody and antigen wasmeasured by the SPR instrument, as a change in refractiveindex over time, and sensorgram data obtained were ana-lyzed using ProteOn Manager TM software.

Competitive ELISA

Competitive ELISA was done to conrm that the S. typhianti-agellin mAbs bind to the same epitope conrmationof recombinant and native agellin. For this, the optimumdilution of the anti-agellin mAbs capable of giving adetectable binding below the saturation level was deter-mined by ELISA using anti-mouse antibody. Recombinantor native S. typhi agellin (0–20 ng ml

- 1 ) was mixed with1:200,000 diluted mAbs (of 1 mg ml

- 1 stocks), incubated(in solution) for 1 h at room temperature, added to antigen-coated wells, and allowed to compete for 1 h at 37 C.Bound mouse antibody was detected by anti-mouse–HRPconjugate (Cell Signaling Technology, Inc., MA, USA).PBS was used as the negative control, and binding of the

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mouse mAb in its presence was taken as the maximumbinding, to calculate the percentage inhibition.

Sandwich ELISA

Sandwich ELISA was done to conrm that the anti-ag-

ellin mAbs could specically capture the different forms(recombinant or native) of soluble S. typhi agellin. Forthis experiment, the optimum concentration of the anti-agellin mAbs was 5 l g ml

- 1 , and two different mAbs,P10F11 and P8F3, were used, for coating 96-well plate(Nunc Maxisorb), by adding 100 l l per well. Puried S.typhi recombinant or native agellins, at different con-centrations (0–1 l g ml

- 1 ), spiked in control human serum(1:100 dilution 1 % BSA solution in PBS, 0.1 % Tween-20), were added to different capture antibody-coated wells(100 l l per well) and incubated for an hour at 37 C. Bi-otinylation of P4D9 and P10F11 anti-agellin mAbs wasperformed by EZ-Link Sulfo-NHS-Biotinylation Kit(Thermo Scientic Pierce, Rockford, IL, USA), whichwere used as detection antibody. Antibody bound to theantigen was detected by anti-biotin–HRP conjugate (CellSignaling Technology, Inc., MA, USA). Control humanserum (1:100 dilution 1 % BSA solution in PBS, 0.1 %Tween-20) was used as the negative control.

Amplication of antibody variable region genes

Total RNA was extracted from hybridoma cells using TRIreagent (Amresco). cDNA was generated by reverse tran-scription Protoscript I RT Kit (New England Biolabs Ltd.Ontario, Canada), using the consensus primers for mouseantibody heavy and light variable region genes [ 35]. ThePCR-amplied V H and V L were cloned, using pGEM -TEasy Vector System I TA cloning kit (Promega Corp.USA), and clones were sequenced. The IMGT/HighV-QUEST analysis tool (available at http://www.imgt.org/ IMGT_vquest/vquest ) was used for genetic diversity ana-lysis of antibody sequences [ 36].

Estimation of cytokines

HEK-hTLR5 and HEK-vector control stable cells (NovusBiologicals, LLC, USA), were cultured in 96-well plates(5 9 105 cells per well), in DMEM media, supplementedwith 10 % (v/v) fetal bovine serum and blasticidin(10 l g ml

- 1 ), at 37 C in 5 % CO 2 . Cells were treated with

the mixture of variable concentrations of native agellin (0,0.5, 1 ng ml

- 1 ) and monoclonal antibody. Supernatantswere collected after 12 h, for cytokine assay. Cytokinelevels were determined using BD OptEIA TM —BD Bio-sciences IL-8 ELISA kit (BD Biosciences San Jose, CA,USA).

Statistical analysis

Student’s t test was used for statistical analysis. p values of \ 0.05 were considered signicant.

Results

Native and recombinant agellin proteinwas puried and characterized

S. typhi iC gene constructs in pPROEXHTb as illustrated inFig. 1, for the expression of recombinant agellin (fulllength; 1-512 AA; * 52 kDa) and hyper-variable middleregion of iC expressing recombinant agellin (middle re-gion; 144-316 AA; * 25 kDa) were used (Fig. 1a, b). Rosetta-gami TM (DE3) E. coli cell lysate conrmed proteinexpression, with majority being in soluble fraction. Afnitypurication yielded pure S. typhi agellin protein, whichwas resolved on 15 % Tris-glycine, SDS-PAGE (Fig. 1c).Similarly, both full-length ( * 52 kDa) and middle-region(* 25 kDa) recombinant agellin proteins of S. typhimuri-um were puried and resolved on 15 % Tris-glycine,SDS-PAGE (Fig. 1c). The yield for all the proteins was* 10 mg l

- 1 ([ 80 % pure) of culture. His-specic im-munoblotting conrmed expression of these proteins(Fig. 1d).

Furthermore, SDS-PAGE analysis showed a single bandof native isolated agellin protein of 52 kDa, for both S.typhi and S. typhimurium , with yields of 0.75 and0.35 mg l

- 1 of culture, respectively (Fig. 1e).

Purication and characterization of S. typhianti-agellin mAbs

Hybridoma technology generated four robust monoclonals,which produced antibody against S. typhi native agellin.Puried monoclonal antibody showed a band of heavy and

Fig. 3 Analysis of stability and antigen-binding strength of anti-agellin mAbs. Effects of destabilizing agents; a KSCN, b pH of buffer c urea d DMSO, and e NaCl, on antigen binding of S. typhianti-agellin mouse monoclonal antibodies. ELISA was done in thepresence of different concentrations of all destabilizing agent. Errorbars represent the standard error calculated from triplicate measure-ments. Competitive ELISA was done to conrm that the anti-agellinmAbs bind to the same antigenic epitope of different forms(f recombinant or g native) of agellin. Different amounts of therecombinant or native agellin were mixed with mAbs, added toantigen-coated (1 l g ml

- 1 ) wells, and allowed to compete for 1 h at37 C. OD values of PBS (negative control)-containing wells wereused to calculate the percentage inhibition

b

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http://www.imgt.org/IMGT_vquest/vquesthttp://www.imgt.org/IMGT_vquest/vquesthttp://www.imgt.org/IMGT_vquest/vquesthttp://www.imgt.org/IMGT_vquest/vquest


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light chains, at their respective molecular weights, whenresolved on 15 % Tris–glycine, SDS-PAGE (Fig. 2a). Thetiters for mAbs were determined by ELISA (Fig. 2b).Antigen-binding property of mAbs was detected by ELISA(in soluble form), Western blot (denatured form), and owcytometry (Fig. 2). Three anti-agellin mAbs (P10F11,P8F3, and P8F10) did not show any cross-reaction with theother Salmonella strains, while one of the mAbs bound tothe native agellin of both S. typhi and S. typhimurium(Fig. 2c, d). Moreover, when recombinant agellin (fulllength) was used for binding analysis of mAbs, it showedsimilar reaction, as shown by native agellins (Fig. 2c, d).However, with the recombinant agellin (middle region),these mAbs showed specic binding to S. typhi only, ex-cept P4D9 mAb, which did not show any binding witheither S. typhi or S. typhimurium (Fig. 2c, d).

Binding analysis of mAbs to agellin attached on bac-terial surface, performed by ow cytometry, showedbinding specicity to S. typhi , for the monoclonals, exceptP4D9 mAb, which was found to bind both with S. typhi andS. typhimurium . None of the four monoclonals exhibitedany cross-reaction with other bacteria (Fig. 2e, f).

Analysis of stability and antigen-binding strengthof anti-agellin mAbs

The binding properties of the anti-agellin mAbs weremore or less comparable among themselves, exceptP10F11 clone, which showed a sturdier binding than theother mAbs, in different conditions. Three S. typhi -specic

mAbs showed extremely stable binding up to 2 M KSCN([ 90 %) and a slight decline in binding at higher concen-tration of KSCN ( [ 70 %), while P4D9 clone showedcomparable stability (up to 65 %), at higher concentrationof KSCN (Fig. 3a). Monoclonals showed similar binding ina pH range of 4–9. Moreover, P8F3 and P8F10 mAbsshowed sharp decline in binding at pH 4. Decline inbinding of P4D9 mAb was observed at pH 2.5, while pHdid not have any effect on binding of P10F11 (Fig. 3b).Furthermore, binding of mAbs in the presence of highconcentrations of urea (up to 6 M), DMSO (up to 10 %),and NaCl (4 M) was observed to be stable and comparable(Fig. 3c–e). Competitive ELISA was performed to conrmthe binding strength of the two sources of S. typhi agellin,native and recombinant, to different mAbs. Recombinant

Fig. 4 Kinetic analysis of the S. typhi native agellin/anti-agellinmouse monoclonal antibody interaction: Sensorgrams for a mAbclone P4D9 (920 RU ligand density), b mAb clone P10F11 (1800 RUligand density), c mAb clone P8F3 (1050 RU ligand density), andd mAb clone P8F10 (1600 RU ligand density), generated in a single-

analyte injection step. Each sensorgram display the response from theve native agellin concentrations: i 100 nM, ii 33.33 nM, iii11.11 nM, iv 3.70 nM, and v 1.23 nM, interacting with one immo-bilization level of mAbs. Straight lines represent the global t of thesensorgrams to a 1:1 kinetic interaction model. RU response unit

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agellin showed varying competitive binding in the solu-

tion, increasing with rising antigen concentration (Fig. 3f).Similarly, native agellin exhibited inhibition but less thanrecombinant antigen (Fig. 3g). This indicated that all themAbs were binding best to the recombinant antigen.

Afnity and diagnostic potential of S. typhianti-agellin mAbs

The afnity interactions between monoclonal antibodies andnative agellin of S. typhi were measured by the SPR in-strument as a change in refractive index over time. Thesensorgram data obtained were tted using a simple 1:1bimolecular interaction model (Langmuir algorithm).Hence, the kinetic parameters such as association (k a or k on ),dissociation (k d or k off ), and equilibrium (K D ) constantsvalue were calculated for binding of Ag, with immobilizedmAbs using the software. The calculated K D values werefound to be in the nanomolar ( C 10

- 9 M) range, for all themAbs, representing their high afnity toward native agellinof S. typhi (Fig. 4). The afnities of P4D9 and P10F11mAbs were highest and comparable and thus of high diag-nostic potential (Fig. 4a, b). P8F3 showed moderate afnitywith a high dissociation rate, while P8F10 had lowest af-nity with a high dissociation rate (Fig. 4c, d; Table 1).Sandwich ELISA was performed with the pair of antibodies(S. typhi anti-agellin capture mAbs and biotinylated anti-agellin detection antibodies), in order to demonstrate theapplication of S. typhi anti-agellin mAbs, for specic de-tection of agellin in the test serum. Pure recombinant full-length or middle-region or native S. typhi agellin in dif-ferent dilutions (0–1 l g ml

- 1 ), spiked in control humanserum (1:100 dilution 1 % BSA solution in PBS, 0.1 %Tween-20), were detected in P10F11 and P8F3 monoclonalantibody-coated wells, using the P4D9 or P10F11

biotinylated anti-agellin mAbs (Fig. 5). Both P10F11 and

P8F3 specically captured recombinant full-length, middle-region, and native agellin in a comparable lower limit, i.e.,* 15 ng ml

- 1 antigen, in the spiked control human serum(Fig. 5a, b). Combination of P8F3 and biotinylated P10F11as capture and detection mAbs exhibited specic detectionof the middle region of S. typhi agellin, spiked in controlhuman serum (Fig. 5a). However, the ELISA pair of P10F11and biotinylated P4D9, as expected, did not generate signalsin the wells containing recombinant middle region of S .typhi agellin, spiked in control human serum (Fig. 5b).

Amplication of variable region of S. typhi

anti-agellin mAbs

The variable regions of both the light (V L ) and heavy (V H )chains, from all the hybridoma clones of S. typhi anti-agellin mouse mAbs, were amplied by RT-PCR, clonedseparately, and sequenced. Sequence alignment of all thefour mAbs depicted 100 % similarity in the variable heavychain, while light chain showed dissimilarity in amino acidsequence of CDR-L1 and CDR-L2 and in the framework region. Germline sequences of mouse monoclonal anti-bodies were identied by searching the InternationalImMunoGeneTics database, to analyze genetic origin, di-versity, and level of maturation of the selected monoclonalantibodies. The four monoclonal antibodies shared asimilar V H germline; IGHV1S56*01F. However, their V Lgermlines were different, showing sequence alignmentwith IGHV (for V H ) and IGKV (for V L ) subgroups(Table 1). The V H and V L regions of mAbs showed re-placement (R) and silent (S) mutations in sequences lo-cated in the complementarity-determining region (CDR)and framework region (FR), which are listed in Table 1.FR showed more R and S mutations than CDR, associated

Table 1 Kinetics and analysis of V region of mouse monoclonal antibodies against S. typhi agellin

Clone Variableregion

Germline(accessionnumber)

No. of mutations

No. of mutations Antigen–antibody interaction (by SPR)

CDR FR K D (M) K a or k on(M

- 1 s- 1 )

K d or k off (s

- 1 )Total CDR/FR R/S R/S

ratioR/S R/S

ratio

P4D9 V L IGKV1-133*01F (Z72382) 13 4/9 3/1 3 8/1 8 5.52E - 10 6.76E ? 04 3.73E - 05

VH IGHV1S56*01 F (M34987) 22 4/18 3/1 3 11/7 1 .57

P10F11 V L IGKV1-117*02 F (M28134) 16 6/10 4/2 2 8/2 4 5.68E - 10 3.53E ? 05 2.01E - 04

VH IGHV1S56*01 F (M34987) 22 4/18 3/1 3 11/7 1 .57

P8F3 V L IGKV6-14*01 F (Y15975) 17 3/12 3/0 ND 12/0 ND 1.02E - 08 3.39E ? 05 3.44E - 03

VH IGHV1S56*01 F (M34987) 22 4/18 3/1 3 11/7 1 .57

P8F10 V L IGKV1-135*01 F (Z72384) 14 6/8 5/1 5 6/2 3 7.88E - 09 3.06E ? 05 2.41E - 03

VH IGHV1S56*01 F (M34987) 23 4/19 3/1 3 11/8 1 .37

SPR surface plasmon resonance, CDR complementarity-determining region, FR framework region, V L variable light chain, V H variable heavychain, R replacement mutations, S silent mutations, ND not determined

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with both V H and V L gene, when sequences of mAbs werecompared to their germline (Table 1).

Demonstration of TLR5 inhibition by S. typhianti-agellin mAbs

To assess whether mAbs can reverse the effect of the

agellin binding to TLR5, which mediate IL-8 secretion, inculture supernatant, we determined the IL-8 cytokinelevels, by adding varying concentration of mAbs in theculture medium, before adding agellin (TLR5 ligand).Out of all S. typhi -specic mAbs, only the P4D9 mAb (atconcentration of 0.5 and 1 l g ml

- 1 ) showed signicantreduction in IL-8 levels, as compared to vector control cells(Fig. 6a). Furthermore, on increasing the concentration of antibody up to 4 l g ml

- 1 , level of IL-8 became similar tothat of vector control (Fig. 6b). This demonstrated, in vitro,

reversal of TLR5-mediated immune response by P4D9mAb, generated on binding to its ligand agellin.

Discussion

Typhoid fever is a signicant global health problem, and its

burden is comparable to some of the diseases prevalentworldwide. However, the current diagnostic modalities forthe typhoid fever have limitations in terms of sensitivity/ specicity and in their approach to universal application.Since the absence of reliable diagnostic methods generallyhampers estimation of the true magnitude and its man-agement, hence, there is undoubtedly a demand for reli-able, simple, and affordable diagnostic test for typhoidfever, particularly in developing countries, where the needis greatest. Possibly the under-explored antigen detection,

Fig. 5 Assessment of the sensitivity of sandwich ELISA for thespecic capture and detection of soluble S. typhi agellin antigenspiked in the control human serum. Microwells were coated using the

optimum concentration of the anti-agellin mouse mAbs (5 l g ml- 1

),P8F3 a or P10F11 b and then reacted with puried soluble S. typhirecombinant or native agellins at different concentrations(0–1 l g ml

- 1 ) spiked in control human serum (1:100 dilution 1 %

BSA solution in PBS, 0.1 % Tween-20). Biotinylated a P10F11 andb P4D9 S. typhi anti-agellin mAbs (1 l g ml

- 1 ) were used asdetection antibody, and the presence of antigen bound to the

antibodies (i.e., ELISA pairs) was detected by anti-biotin–HRPconjugate (Cell Signaling). Control human serum was used as thenegative control. Results are expressed as the mean ± SD opticaldensity at 450 nm

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rather than antibody detection, could provide such a test.Earlier reports suggested detection of S. typhi antigen in theurine of some typhoid patients by co-agglutination [ 37] andELISA [ 38, 39]. However, their specicity varies from 25to 90 %. S. typhi Vi antigen was detected in the urine of most patients with typhoid fever during the rst week of fever onset, when monoclonal Vi capture antibody wasused, in the ELISA, for antigen detection [ 40].

Therefore, through this study, we advocate the use of unique epitopes of S. typhi agellin for the generation andscreening of highly specic mAbs, for developing S. typhi -specic antigen-detection-based immunodiagnostic kits.

For this purpose, to ensure the native conformation of specic epitopes, native agellin ( * 52 kDa) was isolatedfrom a strain of S. typhi (Fig. 1e). Isolated native agellinfrom bacteria was immunogenic and elicited generation of four highly specic monoclonal antibodies, against S. typhiagellin protein. Earlier reports showed isolation of nativeagellin of * 52 kDa from Salmonella culture [ 41–45] andproduction of anti-agellin monoclonal antibodies [ 42, 46 ,47]. We have also isolated genomic DNA, from the clinicalisolate, to perform PCR amplication of iC gene encodingphase 1-d agellin. Expression of full-length (1-512 AA)recombinant S. typhi agellin of * 52 kDa as fusion

Fig. 6 Inhibition of TLR5-mediated immune response by S. typhianti-agellin monoclonal antibodies. IL-8 cytokine was estimated inthe culture supernatant of HEK-hTLR5 stable cells treated withmixture of S. typhi native agellin and anti-agellin monoclonalantibodies. HEK-vector control was used as negative control. a HEK-hTLR5 stimulated with native agellin (0, 0.5, 1 ng ml

- 1 ), and

inhibition by mAbs (at 0, 0.5, 1 l g ml- 1 ) was calculated by

quantitative estimation of IL-8 (pg ml- 1 ). b Inhibition by P4D9

mAb; on increasing the concentration (at 0, 1, 2, 4 l g ml- 1 ), reached

comparable to the basal level of IL-8 secreted, by vector control. Dataare expressed as mean ± SE (pg ml

- 1 ), (* ? p\ 0.05; Student’st test)

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protein along with His-tag was also performed (Fig. 1c).Previously, recombinant agellin as a fusion protein hasbeen expressed using E. coli system [ 42, 43, 48, 49]. In ourcurrent study, we also expressed hyper-variable middleregion of agellin protein (MW: * 25 kDa; 144-316 AA),as a fusion protein (Fig. 1d).

Notably, for highest specicity, we used middle regionof S. typhi agellin protein, for screening of the generatedmonoclonal antibodies. Out of four antibodies generated,three (P10F11, P8F3, and P8F10) showed specicity to S.typhi agellin. However, one (P4D9) was reactive to theagellin of both S. typhi and S. typhimurium (Fig. 2c, d).Conceivably, this cross-reactivity of the pan mAb (P4D9)was due to the contribution of N or C terminus conservedepitopes of agellin protein, since this mAb was unable tobind to the variable middle region of agellin from both S.typhi and S. typhimurium . This phenomenon has also beenreported earlier [ 47]. Additionally, enhanced specicity inantibody detection, by middle region, has been exploitedagainst S. enterica serotype Enteriditis , in poultry [ 50, 51],and in case of S. enterica serovar Brandenburg (S. bran-denburg ) infection, in sheep [ 52].

Monoclonal antibodies were further characterized byELISA and competitive ELISA. Competitive ELISA de-picted exalted binding strength of S. typhi recombinantagellin in the solution, in comparison with the nativeagellin (Fig. 3f, g). Additionally, the binding of mAbs toantigen was highly stable in the presence of differentagents that are conventionally known to destabilize anti-gen–antibody interactions, namely KSCN, DMSO, andurea, by disrupting hydrophobic interaction and NaCl andpH variations which affect electrostatic interactions(Fig. 3a–e). These anti-agellin mAbs had afnity (K d )values C 10

- 9 M, as was observed by SPR technique. Thevery high afnity and stability of these mAbs, produced inthis study, indicate their high antigen binding, therebysignifying their diagnostic potential (Fig. 4; Table 1).Moreover, sequence analysis for somatic mutations (R andS), across the antibody V regions of mouse monoclonalsand their comparison with their germline counterpart, re-veals that these antibodies have evolved and attained theirhigh afnity through antigen-biased somatic hyper-muta-tion phenomenon. It has been reported earlier that an in-crease in afnity and specicity of clones are optimized bysomatic mutations and are antigen-driven processes, whichresults in amplication of positively selected high-afnityclones [ 53, 54]. Thus, our study has generated robustmonoclonal antibodies for specic detection of S. typhiagellin.

Moreover, in this study, we have also analyzed thesemAbs’ ability, to inhibit TLR5–agellin complex. For thispurpose, we have exploited the well-established fact thatTLR5 is activated by its only characterized ligand agellin

[55, 56], using an in vitro system, namely HEK-hTLR5cells, which express TLR5 constitutively and, on stimula-tion with agellin, trigger activation of the transcriptionfactor NF- j B for production of cytokines (IL-8 or IL-6)[57]. It was evident from the results that, as expected, onlypan monoclonal antibody (P4D9), which probably binds toC terminal conserved region of agellin, reversed theTLR5-mediated immune response to a certain extent(Fig. 6). It has been depicted earlier that TLR5 is inducedby binding to the C terminus of the agellin protein [ 56,58]. Therefore, this could possibly help to circumventagellin’s role in regulating virulence and development of Salmonella -induced colitis [ 27, 59] as well as in Crohn’sdisease [ 60].

Furthermore, through this study, we have, for the rsttime, showed binding analysis of these monoclonal anti-bodies to the S. typhi agellin, by ow cytometry, whichdemonstrates the practical possibility of detection of nativeconformation of agellin, present on different bacterialsurface, characterized by higher specicity and no cross-reactivity (Fig. 2e, f). Therefore, these mAbs may be used inthe generation of rapid and real-time systems for the detec-tion of agellated bacteria in contaminated food and there-fore prevent intestinal infections. In the present study,through the sandwich ELISA, we have demonstrated theapplication of these anti-agellin mAbs, in developing a testfor detecting soluble agellin in serum, using P10F11 andP8F3, the two potent anti-agellinmAbs as captureantibodyand biotinylated P4D9 and P10F11 anti-agellin mAbs asdetecting antibodies. This sandwich ELISA appears to bequite sensitive with a detection limit of about 15 ng ml

- 1

(Fig. 5a, b). Our ndings are also in corroboration with aprevious report which used murine mAb-based dual-anti-body sandwich ELISA for rapid detectionof S. typhi agellarantigen in patient serum [ 46].

In conclusion, we have generated a repertoire of robustmonoclonal antibodies against S. typhi agellin, which maybe used in the development of improved diagnostics, par-ticularly in terms of rapidity, sensitivity, and specicity,vis-a-vis the ones currently available. This is in keepingwith the still at-large endemicity and disease burden of typhoid fever, in our country, underlining the importanceof accurate diagnostics, as the mainstay of diseasesurveillance as well as informed policy decisions.

Acknowledgments The authors gratefully acknowledge Dr. AyubQadri (National Institute of Immunology, New Delhi, India) and Dr.Bhabatosh Das (Translational Health Science and Technology Insti-tute), for providing Salmonella strains. This study was supported bygrant from Department of Biotechnology, Government of India, toCentre for Bio-design and Diagnostics, Translational Health Scienceand Technology Institute, and the core grant of Translational HealthScience and Technology Institute to AT. C S was supported by In-novation Award from Centre for Bio-design and Diagnostics.

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Conict of interest The authors declare that they have no conictof interest.

Ethical standards All procedures performed in this study involvinganimals were in accordance with the ethical standards of the institu-tion or practice at the International Centre for Genetic Engineeringand Biotechnology, New Delhi, India.

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a repertoire of high affinity monoclonal antibodies specific to s. typhi

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