research article production and characterization of a

Research ArticleProduction and Characterization of a PolyclonalAntibody of Anti-rLipL21-IgG against Leptospira for EarlyDetection of Acute Leptospirosis

Arivudainambi Seenichamy Abdul Rani BahamanAbdul Rahim Mutalib and Siti Khairani-Bejo

Department of Veterinary Pathology and Microbiology Faculty of Veterinary Medicine Universiti Putra Malaysia43400 Serdang Selangor Malaysia

Correspondence should be addressed to Abdul Rani Bahaman raniupmedumy

Received 7 January 2014 Revised 11 March 2014 Accepted 1 April 2014 Published 22 April 2014

Academic Editor Marcelo A Soares

Copyright copy 2014 Arivudainambi Seenichamy et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Leptospirosis is one of the zoonotic diseases in animals and humans throughout the world LipL21 is one of the importantsurface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen It is widely consideredas a diagnostic marker for leptospirosis In this study we evaluated the serodiagnostic potential of LipL21 protein of Leptospirainterrogans serovar Pomona We have successfully amplified cloned and expressed LipL21 in E coli and evaluated its specificityby immunoblotting Purified recombinant LipL21 (rLipL21) was inoculated into rabbits for the production of polyclonal antibodyCharacterization of the purified IgG antibody against rLipL21 was performed by cross reactivity assay Only sera from leptospirosispatients and rabbit hyperimmune sera recognized rLipL21 while the nonleptospirosis control sera showed no reaction inimmunoblottingWe confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral speciesand it did not react with nonpathogenic leptospires and other bacterial species Results observed showed that anti-rLipL21-IgGantibody has high specificity and sensitivity to leptospires The findings indicated that the antibody could be used in a diagnosticassay for detection of leptospires or their proteins in the early phase of infection

1 Introduction

Leptospirosis is a major public health concern and hasnow been identified as one of the emerging infectiousdiseases worldwide [1 2] It is highly prevalent in the AsiaPacific region [3] In Malaysia leptospiral infections seen indomestic animals were mainly due to serovars of the Sejroeand Pomona serogroups The infections in humans werealso due to Leptospira interrogans serovar Pomona [4] It isconsidered that the majority of leptospirosis cases in humanswere due to association with animals and disease-infectedenvironment [5] Leptospirosis displays a wide array ofclinical presentations and it is difficult to distinguish fromdengue malaria and influenza It mimics many other dis-eases characterized by fever headache andmyalgia [6] Afterclearance of leptospires from blood and body fluids they are

known to persist for prolonged periods in immune privilegedsites like renal tubules brain and eyes of carrier animals [7]Leptospires include 268 serovars [8] and can be organisedinto 31 serogroups on the basis of their antigenic relatedness[5] The antigenic structure of leptospires is complex withstructural heterogeneity in the carbohydrate component ofthe lipopolysaccharides (LPS) [7] The outer membrane ofleptospires is composed of immunogenic LPS and differenttypes of outer membrane proteins (OMPs) Lipopolysac-charides play a key role in immunity to infection and areresponsible for serovar variations [9] Based on serologicalclassification the genus Leptospira was earlier classified intotwo species the pathogenic L interrogans and nonpathogenicL biflexa

The leptospiral membrane possesses at least three types ofOMPs lipoproteins peripheral OMPs and transmembrane

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 592858 8 pageshttpdxdoiorg1011552014592858

2 BioMed Research International

Table 1 Leptospiral strains used in the LipL21 studies

Leptospira Species Serovar Strain

Pathogenic

L interrogans Pomona PomonaL interrogans Canicola Hond Utrecht IVL interrogans Hardjo SponseleeL interrogans Autumnalis Akiyami AL interrogans Djasiman DjasimanL interrogans Grippotyphosa Moskva VL interrogans Hebdomadis HebdomadisL interrogans Javanica Valdrat Batavia 46L interrogans Australis Ballico

L icterohaemorrhagiae Icterohaemorrhagiae RGAL kmetyi Malaysia Bejo-Iso 9119879

Nonpathogenic L biflexa Patoc Patoc 1

proteins [10 11] Among them LipL21 was described asone of the most important outer membrane lipoproteinsproduced during leptospiral infection [12] Detection andidentification of conserved leptospiral lipoproteins amongpathogenic leptospires were made through cross-protectionassays against various serovars of leptospires The exclusivepresence of these proteins in the pathogenic leptospires indi-cated that they could be promising candidates for developingdiagnostics [13] Hence these lipoproteins have currentlybecome a major focus of leptospirosis research [14]

In the present study we expressed the recombinantprotein LipL21 and evaluated its immunogenic potentialwith leptospirosis human sera and rabbit hyperimmune seraWe used purified recombinant LipL21 to create polyclonalimmunoglobulin G (IgG) against rLipL21 The results of thepresent study provide a possible new candidate for devel-oping diagnostic kit for detecting leptospires andor theirleptospiral antigen during the early stages of infection

2 Materials and Methods

21 Bacterial Strains and Growth Conditions In this studyleptospiral reference strains were obtained from WHO Col-laborating Centre Brisbane Queensland Australia as shownin Table 1 Other bacterial strains used in the study includeE coli Pseudomonas aeruginosa Staphylococcus aureus Pas-teurella multocida Bacillus subtilis Salmonella typhi Kleb-siella pneumoniaeand Proteus spp These were kindly pro-vided by the bacteriology Lab Faculty of VeterinaryMedicinein University Putra Malaysia Escherichia coli DH-5120572 (labcollection) and BL21 (DE3) (Novagen Madison WI) wereused for cloning and expression to purify the recombinantprotein Leptospireswere grown tomid-logarithmic phase for7 days at 30∘C in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium The E coli strains were routinelygrown in Luria-Bertani (LB) medium at 37∘C with appro-priate selection pressure (ampicillin (50 120583gmL) kanamycin(50120583gmL) and chloramphenicol (34 120583gmL))

22 Amplification and Cloning of LipL21 Leptospira spp weregrown in EMJH medium to mid-log phase for DNA extrac-tion Genomic DNA extracted from 1 times 108 cells using thePromega Wizard genomic DNA purification kit (PromegaMadison WI USA) The new primers designed were basedon comparison of 56601 sequences retrieved from GenBank(Gene ID 1149354) of L interrogans serovar Lai strain Theprimers used for the amplification of lipL21 gene are FL21-51015840GAG AAGCATATG ATC AAT AGA CTT ATA GC 31015840with restriction site NdeI and RL21-51015840-CCCGAATTC TTATTG TTT GGA AAC CTC TTG-31015840 with restriction siteEcoRI The resulting amplicon was digested with restric-tion enzymes NdeI and EcoRI and inserted into pET-28(b)vector (Novagen) The recombinant plasmid construct wasconfirmed for accurate insertion by both restriction enzymedigestion and sequencing

23 Expression of Recombinant LipL21 Fusion Protein ThepET28-LipL21 (pEL21) plasmid was transformed into Ecoli BL21 (DE3) (Novagen) [15] A single colony of E coliBL21 (DE3) harbouring the pET28-LipL21 (pEL21) plasmidwas inoculated into 10mL LB media containing 50120583gmLkanamycin and incubated overnight at 37∘C with shaking at200 rpm An aliquot of 100 120583L of the overnight cell culturewas added to another tube of 10mL LB medium (containing50 120583gmL kanamycin and Overnight Express AutoinductionSystem 1) (Novagen) and incubated at 20∘C with shaking(200 rpm) The overnight induced culture was harvestedaseptically by centrifugation at 12000 g for 3min E coli BL21(DE3) cells harbouring the pEL21 vector were used as theuninduced or negative control Bacterial pellets recoveredafter inductions were dissolved in appropriate volume ofLaemmli buffer and proteins were resolved on a 12 SDS-PAGE The expression of the recombinant LipL21 (rLipL21)was detected by Western blot using the His tag AP Westernblot kit (Novagen) Protein concentrations were determinedby using a bicinchoninic acid (BCA) protein assay kit (Bio-Rad)

BioMed Research International 3

24 Purification of rLipL21 Fusion Protein For auto induc-tion 1 litre culture was incubated at 20∘C for 18 h withorbital shaking (190 rpm) using the Overnight Express AutoInduction System 1 according to the specifications providedby the manufacturer (Novagen) After the induction periodthe cellswere collected by centrifugation for 10min at 12000 gat 4∘C resuspended in one-tenth of the culture volume ofbinding buffer (50mMNaH

2PO4 300mMNaCl 10mM imi-

dazole pH 80) and subjected to sonication (Branson ultra-sonifier USA) till complete cell lysis The lysates of inducedculture are cleared by centrifugation at 12000 g for 30minat 4∘C and were applied on a His-Trap (Novagen) ofNi2+-nitrilo-triacetic acid (Ni-NTA) affinity column (2mL)Ni-NTA column was preequilibrated with binding buffer(50mM NaH

2PO4 300mM NaCl 10mM imidazole pH

80) and finally it was washed using wash buffer (50mMNaH2PO4 50mMNaCl 10mM imidazole pH80) to remove

the unbound proteins Bound proteins were eluted with elu-tion buffer (50mM NaH

2PO4 300mM NaCl 250mM imi-

dazole pH 60) The eluted recombinant LipL21 protein wasdialysed and concentrated by Centriprep-30 (10 kDa cut off)(Millipore-Amicon Beverly MA)The protein concentrationwas determined byBCAmethodThepurity of LipL21 proteinwas analysed by 12 SDS-PAGE and visualized by stainingwith Coomasie brilliant blue [16]

25 Polyclonal Antibody Production Polyclonal antibodyproductionwas carried out according to themethod of Shanget al [17] Purified rLipL21 protein was loaded onto SDS-12polyacrylamide gel and separated during electrophoresis ArLipL21 containing band was excised from the gel and desic-catedThe desiccated gel containing recombinant protein wasground to a powder dissolved in 1mL of water and mixedwith 1mL of complete Freundrsquos adjuvant (MerckWhitehouseStation NJ) New Zealand White rabbits (free of leptospiralantibodies) were immunized with the mixture of rLipL21 andcomplete adjuvant (subcutaneously and intramuscularly) onDay 1 Additional immunization with the same dosage ofrLipL21 with incomplete Freundrsquos adjuvant (Merck) wasdone on Day 14 Day 28 On Day 42 the rabbits werebled by heart puncture and the serum was tested to detectantibodies against LipL21 The LipL21-antiserum was storedin small aliquots at minus20∘C until use Animals were housed inaccordance with the ethical principles and experimental pro-cedures with animals were approved by the Animal Care andUse Committee of the Faculty of Veterinary Medicine Uni-versity Putra Malaysia (AUP No 09R57Mac 09-Feb10)

26 Purification of IgG fromRabbit Serum To obtain purifiedpolyclonal immunoglobulin whole serum supernatant wasused in Montage Antibody Purification Kits with PROSEP-A(LSK2 ABA 20 Millipore) Immunized rabbit antiserumcontaining IgG was purified according to the manufacturerrsquosinstructions

27 OMPs Separation by Triton X-114 To validate whetherproteins are present in the leptospiral OMPs Triton X-114cellular fractionation was carried out in leptospiral lysates

according to previously described method [14 18] Brieflyleptospires were grown tomid-log phase and the cells washedwith phosphate buffer saline (PBS) containing 5mM MgCl

2

buffer and then resuspended in lysis buffer (1 Triton X-114 containing 150mM NaCl 20mM Tris (pH 8) 2mMEDTA and 1mM phenylmethylsulfonyl fluoride) at 4∘CInsoluble materials of leptospiral OMP were removed bycentrifuging at 17000 g for 10min 20mMCaCl

2was added to

half of the supernatant the supernatant was warmed to 37∘Cand subjected to centrifugation for 10min at 2000 g forphase separation Acetone precipitation was performed toseparate detergent (outer membrane proteins) and aqueous(periplasmic) phases [19]

28 Rabbit Antiserum Rabbit antiserum against Leptospiraspp serovar Australis (strain Ballico) Bataviae (strain Swart)Cynopteri (strain 3522C) Canicola (strain Hond UtrechtIV) Grippotyphosa (strain Moskva V) Hebdomadis (strainHebdomadis) Javanica (Valrat Batavia 46) Pomona (strainPomona) Icterohaemorrhagiae (strain RGA) Tarasovi(strain Perepelistin) Cellodoni (strain Cellodoni) Pyrogenes(strain Salinum) Hardjobovis (strain Sponselee) and Hardjo(strain Hardjoprajito) were obtained from WHO Collab-orating Centre Brisbane Queensland Australia

29 Human Patient Samples Five leptospirosis confirmedpatient serum samples (positive control) and nonlep-tospirosis serum sample which is similar like leptospiro-sis clinical symptoms (negative control) was obtainedanonymously from the Institute for Medical Research(IMR) (httpwwwimrgovmy) Kuala Lumpur Malaysiafor research purpose and it was incorporated into ourresearch project The negative control serum sample men-tioned above was from a nonleptospirosis patent with clini-cally similar symptoms of leptospirosis The serum sampleswere then evaluated by immunoblotting against rLipL21antigens

210 SDS-PAGE and Immunoblotting Leptospires mem-brane fractions or purified proteins of rLipL21 were resolvedon SDS-PAGE The separated proteins were electrotrans-ferred onto a nitrocellulose (NC) membrane (Millipore)Themembrane was blocked with TBS (150mM NaCl 50mMTris-HCl pH 74) containing 005 Tween 20 (T-TBS) and3 bovine serum albumin at 37∘C for 2 hrs After the T-TBSwash the membrane was incubated with primary antibodyovernight and then washed with T-TBS The membranewas then incubated with alkaline phosphatase conjugatedsecondary antibody at 37∘C for 2 hrsThe NCmembrane wasthen developed by BCIP and NBT substrates The LipL21band was visualized by the dark blue colour The enzymereaction on the membrane was terminated by washing withdistilled water [20]

In order to detect recombinant protein of rLipL21the protein was resolved on SDS-PAGE gel The proteinwas then transferred to NC membranes (Millipore) andimmunoblotted using the 1 20000 dilution of primary rab-bit antiserum (against Leptospira spp) and 1 100 dilution


(bp)

1000

500

100

573 bp

M 1 2 3 4 5 6 7 8 9 10 11 12

Figure 1 PCR amplified lipL21 gene from different serovars LaneM 1 kb marker Lane 1 nonpathogenic species Leptospira biflexa(strain Patoc 1) Lane 2 Canicola (strain Hond Utrecht IV) Lane3 Hardjobovis (Sponselee) Lane 4 Autumnalis (strain Akiyami A)Lane 5 Icterohaemorrhagiae (strain RGA) Lane 6 Australis (strainBallico) Lane 7 Pomona (strain Pomona) Lane 8 Grippotyphosa(strainMoskvaV) Lane 9 L kmetyi serovarMalaysia strain Bejo-iso9119879 Lane 10 Balum (strain Mus 127) Lane 11 Grippotyphosa (strainMoskva V) Lane 12 Hebdomadis (strain Hebdomadis)

of human serum (Calbiochem Germany) overnight fol-lowed by T-TBS wash thrice The blot was incubated with1 10000 diluted alkaline phosphatase conjugated secondarygoat anti-rabbit IgG (Calbiochem Germany) or 1 10000diluted alkaline phosphatase conjugated secondary goat anti-human immunoglobulin (Calbiochem Germany) at 37∘C for2 hoursTheNCmembrane (blot) was developed using BCIPand NBT substrates (Novagen USA)

In order to determine if IgG created against the purifiedrecombinant forms of these proteins could effectively bindto their cognate proteins found in the leptospires OMPfraction and other fractions were resolved using SDS-PAGEand transferred to NC membranes These membranes werethen probed using purified anti-rLipL21-IgG at a 1 30000dilution of primary rabbit serum (anti-rLipL21) and goat anti-rabbit IgG-alkaline phosphatase (Calbiochem Germany) ata 1 10000 dilution as a secondary antibody with BCIP andNBT substrates (Novagen USA)

3 Results

31 Amplification of lipL21 Polymerase chain reaction wasperformed using lipL21 gene specific primers for detectionof Leptospira spp The expected size of amplified lipL21gene from pathogenic leptospires strains was 573 bp Noamplification was observed from nonpathogenic leptospires(Figure 1) and other bacterial strains (data not shown)PCR amplification showed that lipL21 gene is conservedamong Leptospira spp only and designed primer specificallyamplified the lipL21 gene from pathogenic Leptospira sppThe DNA sequence was identified analyzed and depositedin the GenBank database (Table 2) Blast analysis showed thatthis sequence is 95 and 96 similar at the nucleotide andpredicted amino acid levels respectively to the sequence of

Table 2 LipL21 gene accession number in the GenBank

Pathogenic strains GenBankL interrogans strain Pomona EU244328L interrogans strain Ballico FJ853169L interrogans strain Hond Utrecht IV FJ853170L interrogans strain Akiyami A FJ853171L interrogans strain Hebdomadis FJ853172L interrogans strain RGA FJ853173L interrogans strain Moskva V FJ853174L interrogans strain Djasiman FJ853175

225

150

100

50

36

25

17

10

(kD

a)

SDS-PAGE Western blot

rLipL21

M U I U I

Figure 2 Expression analysis of rLipL21 on SDS-PAGE andWesternblot Lane U uninduced lysates containing the pEL21 plasmid onlyLane I auto induced lysates containing the pEL21 plasmid Lane Mprotein ladder

annotated gene coding for lipL21 in the complete genomesequence of L interrogans strain Lai

32 Expression and Purification of His-Tagged LipL21 ProteinThe rLip21 gene was cloned in an expression plasmid with anN-terminal His tag and the construct was then transformedinto E coli BL21 His tag was used for the purification ofrecombinant LipL21 (rLipL21) by Ni-NTA affinity columnAliquots of E coli-induced cultures were analyzed on 12SDS-PAGE and expression of rLipL21 protein was confirmedby Western blot with anti-His MAb (Figure 2) The blottedmembrane indicated the presence of corresponding band inthe expressed rLipL21 protein

33 Immunoreactivity of rLipL21 Protein The immunoreac-tivity of the rLipL21 protein against human sera confirmedleptospirosis infection was assessed by immunoblot analysis(Figure 3(b)) and five sera samples from clinically confirmedleptospirosis patients (Figure 3(b)) Figure 3(b) presents


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

(a)

225

150

100

50

36

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9

(b)

Figure 3 (a) Immunoblot reaction of rLipL21 protein recognized bydifferent Leptospira spp in serovar specific hyperimmune sera Lane1 His Tag monoclonal antibody Lane 2 Australis (strain Ballico)Lane 3 Bataviae (strain Swart) Lane 4 Cynopteri (strain 3522C)Lane 5 Canicola (strain Hond Utrecht IV) Lane 6 Grippotyphosa(strain Moskva V) Lane 7 Hebdomadis (strain Hebdomadis) Lane8 Javanica (Valrat Batavia 46) Lane 9 Pomona (strain Pomona)Lane 10 Icterohaemorrhagiae (strain RGA) Lane 11 Tarasovi(strain Perepelistin) Lane 12 Cellodoni (strain Cellodoni) Lane 13Pyrogenes (strain Salinum) Lane 14Hardjobovis (strain Sponselee)Lane 15 Hardjo (strain Hardjoprajito) (b) Immunoblots examiningthe reactivity of leptospirosis positive human sera with rLipL21Lane 1ndash8 05 120583g of purified recombinant rLipL21 was probed withsera from Malaysian leptospirosis patients Lane 1ndash5 leptospirosispatient sera detected rLipL21 and Lane 6ndash9 nonleptospirosis sera(leptospiral like clinical symptoms) not detected rLipL21 Molecularweight standards are indicated in kilodaltons

the typical reaction of serum samples obtained from patientswith (lanes 1 to 5) and without (lanes 6 to 9) leptospiralinfection to detect the rLipL21 The rLipL21 protein withrabbit hyperimmune sera was tested Antibody against thewhole Leptospira spp was detected by immunoblotting inall hyperimmune sera tested with strong signal intensity asobserved (Figure 3(a))

34 Purification of Rabbit Anti-rLipL21 ImmunoglobulinsSDS-PAGE analysis of the purified IgG fraction of the anti-rLipL21 antiserum revealed two bands corresponding to theheavy and light chains of IgG (Figure 4) The protein contentof this fraction was 45mg which was about one-third ofprimary protein content

35 Specificity of Rabbit Anti-rLipL21-IgG Antibody IgG-enriched fractions (anti-rLipL21-IgG) were able to recognize

225

150

100

50

36

25

17

10

(kD

a)

L FT W 1 2 3

H-chain 50kDa

L-chain 22kDa

Figure 4 Purification of (anti-rLipL21) IgG antibody was purifiedfrom rabbit antiserum against rLipL21 Lane L anti-rLipL21 serumLane FT flow through of unbound proteins from column LaneW wash of nonspecific proteins from column Lane 1 elutedfraction without protein Lane 2 purified anti-rLipL21-IgG Lane3 concentrated anti-rLipL21-IgG Molecular weight standards areindicated in kilodaltons

225150100

5036

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9 10 11 12

Figure 5 Immunoblot of panel of Leptospira spp obtained by usingrabbit anti-rLipL21-IgG antibody to detect single band in leptospiralLipL21 antigen in detergent phase Lane 1 Canicola (strain HondUtrecht IV) Lane 2 Hardjobovis (Sponselee) Lane 3 Autumnalis(strainAkiyamiA) Lane 4 Icterohaemorrhagiae (strainRGA) Lane5 Australis (strain Ballico) Lane 6 Pomona (strain Pomona) Lane7 Grippotyphosa (strain Moskva V) Lane 8 L kmetyi serovarMalaysia strain Bejo-iso 9119879 Lane 9 Balum (strain Mus 127) Lane10 Grippotyphosa (strain Moskva V) Lane 11 Hebdomadis (strainHebdomadis) Lane 12 nonpathogenic species L biflexa (strainPatoc 1) Molecular weight standards are indicated in kilodaltons

leptospiral native LipL21 antigen by immunoblot whichdemonstrated that the antibody was specific against LipL21from pathogenic Leptospira spp (Figure 5) Preimmuneserum did not react with the rLipL21 proteins (data notshown) When these antibodies were tested against triton X-114 fraction of pathogenic serovars of Leptospira spp theybound to proteins that corresponded to the molecular weight


of the native protein From this result it can be assumed thatthe recombinant forms of the candidate antigens effectivelymimic the properties of the native form

4 Discussion

Leptospirosis is an important public health disease inendemic areas all over the world In Malaysia continuouspresence of multiple leptospires serovars is observed incompetent reservoirs like rat and other animals and a largepopulation of susceptible hosts [4] Humans and animalsinfected with leptospires mount strong and rapid antibodyresponses directed against the outer surface protein LipL21The presence of LipL21-directed antibodies in patient serumis a reliable marker of leptospiral infection Lipoproteins arean important antigen and play a key role in the pathogenesisof leptospirosis [14] and surface-exposed putative lipopro-teins [21 22] Several lipoproteins and surface-exposed puta-tive lipoproteins have been identified in Leptospira sppwhich are associated with the outer membrane particularlythose exposed on the cell surface where bacterial pathogensinteractwith the host [23]These lipoproteins aremaintainingthe bacterial cell structure attachment to various substratesand immunogenicity [5] Among these lipoproteins theLipL21 protein has been well characterized in L interrogansserovar Lai [24] LipL21 has been reported to be conservedin pathogenic leptospiral strains and this has led to its usein PCR-based identification of Leptospira spp [7] In thisstudy a PCR assay was performed using lipL21 gene specificprimers for detection of Leptospira spp The expected size(573 bp) of amplified lipL21 gene was seen in 11 pathogenicLeptospira strains but not observed in the nonpathogenicleptospiral strain (Figure 1) Similarly the lipL21 gene specificprimer did not amplify any other 10 bacterial strains (data notshown) The lipL21 gene specific primer would be useful foridentification of pathogenic Leptospira sppThese PCR resultscorroboratedwell with previous reports that LipL21 is presentonly in pathogenic and absent in nonpathogenic strains [1225]

Genomic analysis revealed that the identified LipL21proteins of L interrogans strain Pomonawere found to be 97ndash100 amino acids similar to other pathogenic Leptospira sppAn alignment of the LipL21 sequence from pathogenic Lep-tospira revealed 96 to 100 identity They also did not haveany significant similarity with the proteins of other organisms[26 27] However insights from recent genome projects haveshown that nonpathogenic strain of L biflexa LipL21 proteinis an ortholog with sim50 identity present in pathogenicleptospires [26] Its exclusive presence in pathogenic lep-tospires indicated that this protein could be a goodcandidate in developing diagnostic kits [13]

Generally important diagnostic and vaccinemarkers pri-marily induce antibodies against surface structures Molec-ular analysis of leptospiral OMP antigens is important inunderstanding the antibody response of hosts because theantigenic diversity may influence the specificity and sensitiv-ity of the serological assay The conserved nature and highlevel of expression of LipL21 among pathogenic Leptospiraspp [7] suggest that rLipL21 immunoblot may show similar

performance regardless of the local isolates E coli basedexpression system is now routinely used for the synthesis ofrecombinant proteins for a variety of purposes ranging fromstructural studies to the development of vaccines [11 12 19]Similarly the purification of recombinant protein is impor-tant to develop a detection system for infectious diseasesThe present work adopted similar approach for expressionof LipL21 protein and purification of rLipL21 in E colicells (Figure 2)

Normally E coli based IPTGNaCl induced expressionsystemwas used for the synthesis of recombinant lipoproteinsfrom Leptospira spp [28ndash30] Drawback for the use of thissystem includes the requirement of expression conditionssuch as need to follow growth of bacterial culture and addIPTGNaCl at the proper time An advantage of the use ofOvernight Auto Induction System 1 in the present study didnot require any inducer including IPTGNaCl

In this study an immunoblot using the rLipL21 proteinas an antigen was evaluated for the diagnosis of leptospirosisin humans In this study we evaluated the clinical utility andthe corresponding sensitivity and specificity of recombinantLipL21 based immunoblot for the serodiagnosis of leptospiro-sis The specificity of the antibody test (immunoblot) with arLipL21 protein detected by clinically confirmed leptospirosispatient sera (Figure 3(b)) In order to confirm the similarityof the antigenic structure of the recombinant forms of theLipL21 protein as well as to verify the cell wall localizationof LipL21 we carried out a simple immunoblot study inwhich we tested the ability of the recombinant proteins toreact with serum from a rabbit (Figure 3(b)) The strongreactivity of the recombinant forms of LipL21 with the patientserum confirms the information from studies carried outpreviously [12 31] Recombinant LipL21was also reactivewiththis serum meaning that LipL21 is immunogenic during lep-tospiral infection This is probably because LipL21 protein isunique and highly conserved to pathogenic Leptospira spp[12] The specificity of LipL21 has been demonstrated tobe strong immunogenic and could be an efficient tracer toLipL21 identification from Leptospira spp Thus use of therLipL21 antigen in immunoblot has the potential to becomea useful tool for serodiagnosis of leptospiral infection

Lipoproteins may be used as novel targets for the devel-opment of infection markers and leptospirosis vaccines [5]In pathogenic Leptospira spp LipL21 has been demonstratedto be a strong immunogen which had been considered forvaccine development [12 27 29] Together these findingssuggest that rLipL21 antigen is specific and sensitive for thedetection of antibodies against leptospiral infection Bothrabbit and human anti-leptospiral antibodies were found tobe strongly reactive with rLipL21 Additional immunoblotresults showed that the rLipL21 had the advantage of highspecificity and sensitivity to leptospirosis The present studyfollows the strategy of previous studies on rLipL32 rOmpL1rLipL41 and rLigA as target antibodies used for clinicaldiagnosis of leptospirosis [32ndash35]

Although many of the cellular proteins were insoluble inthe Triton X-114 or remained present in the aqueous phasea limited number of proteins were present in the detergentphase as previously described by Cullen et al [12] and


Haake et al [28]However LipL21 proteinwas observed in theinsoluble detergent phase and not observed in soluble phase[12] Regardless of this when we performed the conversestudy the detergent fraction containing OMP antigen fromLeptospira spp and sera of immunized rabbit reacted againstLipL21 antigen Immunoblot of OMP fraction of Leptospiraspp using anti-rLipL21-IgG antibody revealed specificityof LipL21 antigen from pathogenic strains of leptospires(Figure 5) OMP fractions from the Leptospira spp wereLipL21 bound strongly detected by their anti-rLipL21-IgGantibodies This result revealed that LipL21 is localizedon the outer portion of the leptospires This is the firstdemonstration of experimental characterization of the LipL21from L interrogans strain Pomona in Malaysia Results fromthis study suggested that rLipL21 is a potential candidatefor the serodiagnosis of leptospirosis Further research isrequired to evaluate the effectiveness of this antigen witha larger number of samples obtained during outbreaks ofleptospirosis The polyclonal serum against rLipL21 proteinwasmore specific for differentiating the pathogenic organismfrom nonpathogenic leptospires Apart from these our workrevealed that the use of anti-rLipL21-IgG antibodies increasesthe specificity of the antigen detection From these results weconclude that the use of purified recombinant protein basedantibody production is an appropriate and applicablemethodfor detection of acute leptospirosis

The polyclonal antibodies were highly sensitive but lessspecific in comparison to monoclonal antibodies Mono-clonal antibodies on the other hand were highly specific butless sensitive [36]These data could have useful application todetecting LipL21 antigen from leptospires infection

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the technical assistance of MrMohd Azri Roslan from the Department of VeterinaryPathology andMicrobiology Faculty of VeterinaryMedicineUniversity Putra Malaysia This work was supported by theScience Fund Grant no 02-01-04 SF 0199 from the Ministryof Science Technology and Innovation (MOSTI) Malaysia

References

[1] S Faine B Adler C A Bolin and P Perolat Leptospira andLeptospirosis Medisci Press Melbourne Australia 2nd edition1999

[2] A R Bharti J E Nally J N Ricaldi et al ldquoLeptospirosisa zoonotic disease of global importancerdquo Lancet InfectiousDiseases vol 3 no 12 pp 757ndash771 2003

[3] A F B Victoriano L D Smythe N Gloriani-Barzaga et alldquoLeptospirosis in the Asia Pacific regionrdquo BMC InfectiousDiseases vol 9 article 147 2009

[4] B Siti-Khairani Epidemiology of Leptospira interrogans SerovarHardjo Infection in Cattle [PhD thesis] University PutraMalaysia Selangor Malaysia 2001

[5] P N Levett ldquoLeptospirosisrdquo Clinical Microbiology Reviews vol14 no 2 pp 296ndash326 2001

[6] T K Koay S Nirmal L Noitie and E Tan ldquoAn epidemiologicalinvestigation of an outbreak of leptospirosis associated withswimming Beaufort Sabahrdquo Medical Journal of Malaysia vol59 no 4 pp 455ndash459 2004

[7] A I Ko C Goarant andM Picardeau ldquoLeptospira the dawn ofthe molecular genetics era for an emerging zoonotic pathogenrdquoNature Reviews Microbiology vol 7 no 10 pp 736ndash747 2009

[8] D J Brenner A F Kaufmann K R Sulzer A G SteigerwaltF C Rogers and R S Weyant ldquoFurther determination ofDNA relatedness between serogroups and serovars in the familyLeptospiraceae with a proposal for Leptospira alexanderi spnov and four new Leptospira genomospeciesrdquo InternationalJournal of Systematic Bacteriology vol 49 no 2 pp 839ndash8581999

[9] A de la Pena-Moctezuma D M Bulach and B Adler ldquoGeneticdifferences among the LPS biosynthetic loci of serovars ofLeptospira interrogans and Leptospira borgpeterseniirdquo FEMSImmunology and Medical Microbiology vol 31 no 1 pp 73ndash812001

[10] P A Cullen D A Haake and B Adler ldquoOuter membrane pro-teins of pathogenic spirochetesrdquo FEMS Microbiology Reviewsvol 28 no 3 pp 291ndash318 2004

[11] P A Cullen X Xu J Matsunaga et al ldquoSurfaceome ofLeptospira spprdquo Infection and Immunity vol 73 no 8 pp 4853ndash4863 2005

[12] P A Cullen D A Haake D M Bulach R L Zuerner andB Adler ldquoLipL21 is a novel surface-exposed lipoprotein ofpathogenic Leptospira speciesrdquo Infection and Immunity vol 71no 5 pp 2414ndash2421 2003

[13] Z Wang L Jin and A Wegrzyn ldquoLeptospirosis vaccinesrdquoMicrobial Cell Factories vol 6 article 39 2007

[14] M Pinne and D A Haake ldquoA comprehensive approach toidentification of surface-exposed outer membrane-spanningproteins of Leptospira interrogansrdquo PLoS ONE vol 4 no 6Article ID e6071 2009

[15] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor NY USA 2001

[16] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[17] E S Shang T A Summers and D A Haake ldquoMolecularcloning and sequence analysis of the gene encoding LipL41 asurface-exposed lipoprotein of pathogenic Leptospira speciesrdquoInfection and Immunity vol 64 no 6 pp 2322ndash2330 1996

[18] D A Haake E M Walker D R Blanco C A Bolin J NMiller and M A Lovett ldquoChanges in the surface of Leptospirainterrogans serovar grippotyphosa during in vitro cultivationrdquoInfection and Immunity vol 59 no 3 pp 1131ndash1140 1991

[19] R L Zuerner W Knudtson C A Bolin and G TruebaldquoCharacterization of outer membrane and secreted proteins ofLeptospira interrogans serovar pomonardquoMicrobial Pathogenesisvol 10 no 4 pp 311ndash322 1991


[20] C G OrsquoConnor and L K Ashman ldquoApplication of the nitro-cellulose transfer technique and alkaline phosphatase conju-gated anti-immunoglobulin for determination of the specificityof monoclonal antibodies to protein mixturesrdquo Journal ofImmunological Methods vol 54 no 2 pp 267ndash271 1982

[21] D D Hartwig F K Seixas G M Cerqueira A J A McBrideand O A Dellagostin ldquoCharacterization of the immunogenicand antigenic potential of putative lipoproteins from Leptospirainterrogansrdquo Current Microbiology vol 62 no 4 pp 1337ndash13412011

[22] W Viratyosin S Ingsriswang E Pacharawongsakda and PPalittapongarnpim ldquoGenome-wide subcellular localization ofputative outer membrane and extracellular proteins in Lep-tospira interrogans serovar Lai genome using bioinformaticsapproachesrdquo BMC Genomics vol 9 article 181 2008

[23] J C Setubal M Reis J Matsunaga and D A Haake ldquoLipopro-tein computational prediction in spirochaetal genomesrdquoMicro-biology vol 152 no 1 pp 113ndash121 2006

[24] H He W Wang Z Wu Z Lv J Li and L Tan ldquoProtection ofguinea pigs against Leptospira interrogans serovar Lai by lipL21DNA vaccinerdquoCellular andMolecular Immunology vol 5 no 5pp 385ndash391 2008

[25] P S Cheema S K Srivastava R Amutha S Singh H SinghandM Sandey ldquoDetection of pathogenic leptospires in animalsby PCR based on lipL21 and lipL32 genesrdquo Indian Journal ofExperimental Biology vol 45 no 6 pp 568ndash573 2007

[26] M Picardeau ldquoConjugative transfer between Escherichia coliand Leptospira spp as a new genetic toolrdquoApplied and Environ-mental Microbiology vol 74 no 1 pp 319ndash322 2008

[27] H-L Yang Y-Z Zhu J-H Qin et al ldquoIn silico andmicroarray-based genomic approaches to identifying potential vaccinecandidates against Leptospira interrogansrdquo BMC Genomics vol7 article 293 2006

[28] DAHaakeGChao R L Zuerner et al ldquoThe leptospiralmajorouter membrane protein LipL32 is a lipoprotein expressedduring mammalian infectionrdquo Infection and Immunity vol 68no 4 pp 2276ndash2285 2000

[29] D A Haake C I Champion C Martinich et al ldquoMolecularcloning and sequence analysis of the gene encoding OmpL1a transmembrane outer membrane protein of pathogenic Lep-tospira spprdquo Journal of Bacteriology vol 175 no 13 pp 4225ndash4234 1993

[30] J Matsunaga K Werneid R L Zuerner A Frank and DA Haake ldquoLipL46 is a novel surface-exposed lipoproteinexpressed during leptospiral dissemination in the mammalianhostrdquoMicrobiology vol 152 no 12 pp 3777ndash3786 2006

[31] D Luo F Xue DMOjcius et al ldquoProtein typing ofmajor outermembrane lipoproteins from Chinese pathogenic Leptospiraspp and characterization of their immunogenicityrdquo Vaccinevol 28 no 1 pp 243ndash255 2009

[32] S Dey C M Mohan T M A S Kumar P Ramadass A MNainar and K Nachimuthu ldquoRecombinant LipL32 antigen-based single serum dilution ELISA for detection of canineleptospirosisrdquo Veterinary Microbiology vol 103 no 1-2 pp 99ndash106 2004

[33] K Natarajaseenivasan P Vijayachari S Sharma A P SugunanJ Selvin and S C Sehgal ldquoSerodiagnosis of severe leptospirosisevaluation of ELISA based on the recombinant OmpL1 orLipL41 antigens of Leptospira interrogans serovar autumnalisrdquo

Annals of Tropical Medicine and Parasitology vol 102 no 8 pp699ndash708 2008

[34] R U M Palaniappan Y-F Chang S S D Jusuf et al ldquoCloningand molecular characterization of an immunogenic LigA pro-tein of Leptospira interrogansrdquo Infection and Immunity vol 70no 11 pp 5924ndash5930 2002

[35] M Subathra T M A Senthilkumar G L Ramya and PRamadass ldquoEvaluation of a novel IGG-ELISA for serodiagnosisof leptospirosis using recombinant LipL32 antigen of L Inter-rogans serovar icterohaemorrhagiae in dogsrdquo Asian Journal ofMicrobiology Biotechnology and Environmental Sciences vol 10no 2 pp 445ndash447 2008

[36] M Varma M Morgan B Jasani P Tamboli and M B AminldquoPolyclonal anti-PSA is more sensitive but less specific thanmonoclonal anti-PSA implications for diagnostic prostaticpathologyrdquo American Journal of Clinical Pathology vol 118 no2 pp 202ndash207 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Leptospiral strains used in the LipL21 studies

Leptospira Species Serovar Strain

Pathogenic

L interrogans Pomona PomonaL interrogans Canicola Hond Utrecht IVL interrogans Hardjo SponseleeL interrogans Autumnalis Akiyami AL interrogans Djasiman DjasimanL interrogans Grippotyphosa Moskva VL interrogans Hebdomadis HebdomadisL interrogans Javanica Valdrat Batavia 46L interrogans Australis Ballico

L icterohaemorrhagiae Icterohaemorrhagiae RGAL kmetyi Malaysia Bejo-Iso 9119879

Nonpathogenic L biflexa Patoc Patoc 1

proteins [10 11] Among them LipL21 was described asone of the most important outer membrane lipoproteinsproduced during leptospiral infection [12] Detection andidentification of conserved leptospiral lipoproteins amongpathogenic leptospires were made through cross-protectionassays against various serovars of leptospires The exclusivepresence of these proteins in the pathogenic leptospires indi-cated that they could be promising candidates for developingdiagnostics [13] Hence these lipoproteins have currentlybecome a major focus of leptospirosis research [14]

In the present study we expressed the recombinantprotein LipL21 and evaluated its immunogenic potentialwith leptospirosis human sera and rabbit hyperimmune seraWe used purified recombinant LipL21 to create polyclonalimmunoglobulin G (IgG) against rLipL21 The results of thepresent study provide a possible new candidate for devel-oping diagnostic kit for detecting leptospires andor theirleptospiral antigen during the early stages of infection

2 Materials and Methods

21 Bacterial Strains and Growth Conditions In this studyleptospiral reference strains were obtained from WHO Col-laborating Centre Brisbane Queensland Australia as shownin Table 1 Other bacterial strains used in the study includeE coli Pseudomonas aeruginosa Staphylococcus aureus Pas-teurella multocida Bacillus subtilis Salmonella typhi Kleb-siella pneumoniaeand Proteus spp These were kindly pro-vided by the bacteriology Lab Faculty of VeterinaryMedicinein University Putra Malaysia Escherichia coli DH-5120572 (labcollection) and BL21 (DE3) (Novagen Madison WI) wereused for cloning and expression to purify the recombinantprotein Leptospireswere grown tomid-logarithmic phase for7 days at 30∘C in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium The E coli strains were routinelygrown in Luria-Bertani (LB) medium at 37∘C with appro-priate selection pressure (ampicillin (50 120583gmL) kanamycin(50120583gmL) and chloramphenicol (34 120583gmL))

22 Amplification and Cloning of LipL21 Leptospira spp weregrown in EMJH medium to mid-log phase for DNA extrac-tion Genomic DNA extracted from 1 times 108 cells using thePromega Wizard genomic DNA purification kit (PromegaMadison WI USA) The new primers designed were basedon comparison of 56601 sequences retrieved from GenBank(Gene ID 1149354) of L interrogans serovar Lai strain Theprimers used for the amplification of lipL21 gene are FL21-51015840GAG AAGCATATG ATC AAT AGA CTT ATA GC 31015840with restriction site NdeI and RL21-51015840-CCCGAATTC TTATTG TTT GGA AAC CTC TTG-31015840 with restriction siteEcoRI The resulting amplicon was digested with restric-tion enzymes NdeI and EcoRI and inserted into pET-28(b)vector (Novagen) The recombinant plasmid construct wasconfirmed for accurate insertion by both restriction enzymedigestion and sequencing

23 Expression of Recombinant LipL21 Fusion Protein ThepET28-LipL21 (pEL21) plasmid was transformed into Ecoli BL21 (DE3) (Novagen) [15] A single colony of E coliBL21 (DE3) harbouring the pET28-LipL21 (pEL21) plasmidwas inoculated into 10mL LB media containing 50120583gmLkanamycin and incubated overnight at 37∘C with shaking at200 rpm An aliquot of 100 120583L of the overnight cell culturewas added to another tube of 10mL LB medium (containing50 120583gmL kanamycin and Overnight Express AutoinductionSystem 1) (Novagen) and incubated at 20∘C with shaking(200 rpm) The overnight induced culture was harvestedaseptically by centrifugation at 12000 g for 3min E coli BL21(DE3) cells harbouring the pEL21 vector were used as theuninduced or negative control Bacterial pellets recoveredafter inductions were dissolved in appropriate volume ofLaemmli buffer and proteins were resolved on a 12 SDS-PAGE The expression of the recombinant LipL21 (rLipL21)was detected by Western blot using the His tag AP Westernblot kit (Novagen) Protein concentrations were determinedby using a bicinchoninic acid (BCA) protein assay kit (Bio-Rad)














2


2was added to







(bp)

1000

500

100

573 bp

M 1 2 3 4 5 6 7 8 9 10 11 12




3 Results




225

150

100

50

36

25

17

10

(kD

a)


rLipL21

M U I U I






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

(a)

225

150

100

50

36

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9

(b)





225

150

100

50

36

25

17

10

(kD

a)

L FT W 1 2 3

H-chain 50kDa

L-chain 22kDa


225150100

5036

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9 10 11 12





4 Discussion














Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























2


2was added to







(bp)

1000

500

100

573 bp

M 1 2 3 4 5 6 7 8 9 10 11 12




3 Results




225

150

100

50

36

25

17

10

(kD

a)


rLipL21

M U I U I






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

(a)

225

150

100

50

36

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9

(b)





225

150

100

50

36

25

17

10

(kD

a)

L FT W 1 2 3

H-chain 50kDa

L-chain 22kDa


225150100

5036

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9 10 11 12





4 Discussion














Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(bp)

1000

500

100

573 bp

M 1 2 3 4 5 6 7 8 9 10 11 12




3 Results




225

150

100

50

36

25

17

10

(kD

a)


rLipL21

M U I U I






1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

(a)

225

150

100

50

36

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9

(b)





225

150

100

50

36

25

17

10

(kD

a)

L FT W 1 2 3

H-chain 50kDa

L-chain 22kDa


225150100

5036

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9 10 11 12





4 Discussion














Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

(a)

225

150

100

50

36

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9

(b)





225

150

100

50

36

25

17

10

(kD

a)

L FT W 1 2 3

H-chain 50kDa

L-chain 22kDa


225150100

5036

25

17

10

(kD

a)

1 2 3 4 5 6 7 8 9 10 11 12





4 Discussion














Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4 Discussion














Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article production and characterization of a

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