Peptide specific monoclonal antibodies of Leptospiral LigA for acute diagnosis of leptospirosis

Leptospirosis is underdiagnosed due to low sensitivity, need of specialised equipment, and expensive reagents for serological and molecular diagnosis respectively. Considering the sensitivity, rapidity, inexpensive reagents and collection of clinical samples, the monoclonal antibody based antigen detection method from urine samples has been developed and evaluated. LigA (LK90) based B-cell specific epitopes were predicted and synthesised as peptides for the production of monoclonal antibody. LK90543: SNAQKNQGNA (amino acids: 543 to 552), and LK901110: DHHTQSSYTP (amino acids: 1110 to 1119) with VaxiJen score of 1.3719 and 1.2215, respectively were used. Thirty two and 28 urine samples from confirmed and seronegative healthy human subjects, respectively were included for the evaluation of MAb-based dot blot ELISA. The specificity of the evaluated MAbs, P1B1 and P4W2 were found to be in the range of ~93–96%. Moreover, the MAbs did not show cross-reactivity with other bacterial antigens as confirmed by IgG ELISA, further validating its specificity for leptospiral antigens. These findings suggest that the developed MAb based dot blot ELISA is a simple, rapid performed in less than 8 h, inexpensive with a ICER of $8.7/QALY, and affordable in developing countries and area where laboratory facilities are limited.

effective solution to this difficulty 9 . Assays for the detection of leptospiral antigens and DNA are still being developed 10 , including conventional and real-time PCR assays. However, the molecular methods are unaffordable due to the need for specialized equipment and expensive reagents that are not available during outbreak situations in the field and for routine diagnosis.
MAb based dot-blot assay (antigen capture) could positively be an inexpensive, rapid, and easy to perform diagnostic assay. With this view in the present study we have computationally identified immunogenic epitopes of LK90 (LigA-90 kDa) that can bind B-cells and provoke immune responses. The predicted peptides were synthesized, used to immunize mice and develop monoclonal antibodies that can be utilized in the development of novel antigen detection immunodiagnostic assays. The MAbs obtained were able to detect the native proteins in several leptospiral serovars as evidenced by Western blotting and IgG ELISA. The dot-blot ELISA developed with these MAbs were found to be highly specific for conclusive diagnosis of acute leptospirosis during outbreak situations.
A panel of bacterial pathogens other than Leptospira including Escherichia coli, Staphylococcus aureus, Serratia marcescens, Citrobacter freundi and Pseudomonas aeruginosa was included as antigens to study the specificity of the developed MAbs. All the bacterial isolates were maintained in Luria-Bertani (LB) agar by regular sub-culturing.

Study site, Patients, case definition and ethics.
The samples recruited for this study were through our routine hospital based surveillance at Annal Mahatma Gandhi Memorial General Hospital, Tiruchirappalli, Tamilnadu, India. In total 32 urine and serum samples from laboratory confirmed leptospirosis cases (a positive IgM ELISA, isolation of leptospires from the blood, seroconversion or four-fold rise in titre by MAT) were collected during the early phase of illness (between 0 and 10 days; Table S1). The age, and sex wise distribution of the confirmed cases included in the study are given (Table S2). A total of 28 seronegative healthy controls matched with respect to age (±5 years) and sex, 28 patients who were hospitalized with a clinical suspicion of leptospirosis and subsequently diagnosed as having other illness based on laboratory evidence, and 25 patients confirmed for Syphilis (13) and Lyme diseases (12) were also included to study the efficiency of the dot-blot ELISA developed in the present study. Informed written consent was obtained from both cases and controls before sampling, and the study protocol was approved by the Institutional Ethics Committee (IEC) of Bharathidasan University (DM/2007/101/373/ Project No. 2) as well as permitted by the Directorate of Health Services (Ref. No. 5796/ TV 1/07), Tamilnadu. The methods were carried out in accordance with the relevant guidelines and regulations.
Rodents were trapped from the same locality of human cases of Tiruchirapalli district. Trap cages were used and additional captures were conducted by locales 12 . A total of 33 urine and serum samples were obtained from field rats and are also included in the study.
Two groups of BALB/c mice were experimentally infected with different serovars of Autumnalis (Group A; 11 animals each infected separately with L. interrogans serovar Autumnalis strains N2, Akiyami, and Bangkinang) or EMJH media (Group B). Seven to ten days after infection, serum and urine were collected aseptically. The Institutional Animal Ethics Committee (IAEC) of Bharathidasan University (BDU/IAEC/25/2013/09.04.2013) approved the study protocols. The methods were carried out in accordance with the relevant guidelines and regulations.
Sample processing and storage. Venous blood samples were collected in tubes without anticoagulant and transported to laboratory on ice. Serum was separated, frozen in aliquots at −80 °C and used to perform MAT and IgM ELISA with live and heat extracted leptospiral antigens respectively for laboratory confirmation of leptospirosis. Individual urine samples from human, rodents and experimentally infected mice were centrifuged at 12,000 × g for 10 min at room temperature; the supernatant was discarded leaving an aliquot of 50 μl, boiled for 30 min and centrifuged at 1,000 × g for 10 minutes. The supernatant obtained was subjected for protein estimation by bicinchoninic acid (BCA) method (Sigma-Aldrich, St. Louis, MO) and desired concentration was dotted individually onto NC strips.
Microscopic agglutination test. Serum samples from patients and rodents were used for MAT. MAT was performed with the panel of 13 leptospiral serovars. Seven days old culture at a concentration of 1-2 × 10 8 organisms/ml was used as antigen in MAT. Each serum was tested in doubling dilutions starting from 1:20. MAT titers were reported as the reciprocal of the dilution agglutinating ≥50% of live bacterial antigen and a titre of ≥1:80 was considered positive. India being a tropical country and endemic for leptospirosis, a MAT titer of ≥1 in 80 was considered as a cut-off titre for MAT positivity 14, 15 . Mouse adapted challenge strains. Mouse Adapted Challenge Strains (MACS) were selected from their corresponding parent strains (reference laboratory strains), by passaging them in BALB/c mice (cyclophosphamide treated) for ~15 times. The MACS strains were passaged twice in vitro and used for preparation of heat extracted antigens 13 . Preparation of heat extracted antigens. Leptospiral heat extracted antigen was prepared from 7 days old well-grown leptospiral culture (1-2 × 10 8 /ml) of MACS. The cultures were killed by formalin (final concentration 0.5%, v/v), heated in a boiling water bath for 30 min and centrifuged for 30 min at 10,000 × g. The supernatants were used as heat extracted antigens. Protein concentrations were determined by BCA method (Sigma-Aldrich, St. Louis, MO).
Prediction of LigA specific B-cell epitopes. The protein sequence of LK90 with accession numbers AAB53736 was retrieved from GenBank and subjected for B-cell epitope prediction using BCPred 15 . B-cell epitopes having BCPred score >0.9 and VaxiJEN score >0.4 were selected for further study.
Peptide synthesis and purification. The peptides that meet the criteria to be highly immunogenic were synthesized by the solid-phase method using 9-fluorenylmethoxy carbonyl (Fmoc) chemistry with biotin at the N-terminus, linked to the peptide sequence through a spacer sequence of "SGSG" and keyhole limpet hemocyanin (KLH) as a carrier protein conjugated at the C-terminus (Peptide 2.0, Inc., USA). The purity of each peptide was ~95% by analytical RP-HPLC and mass spectrometry.
Immunization and hybridoma production. Six week old BALB/c mice were injected intraperitoneally with 50 µg of synthetic peptides on days 0, 14, 21 and 28. Freund's complete adjuvant was used for the first dose of immunization and incomplete for the subsequent doses. Three days after the last booster, titre was checked by IgG ELISA. Splenic lymphocytes obtained from mice with the highest ELISA titre were fused with murine SP2/0-Ag14 myeloma cells (NCCS, Pune) in the presence of PEG-1450 (Sigma-Aldrich, St. Louis, MO). The hybridomas were selected in HAT medium and screened for the production of desired antibodies with an IgG ELISA using homologous antigens. Positive hybridoma cells were cloned using limiting dilution to obtain antibodies from a single cell. Purification of antibodies were achieved by ammonium sulfate precipitation of culture supernatant, followed by affinity chromatography using a Pierce Protein G Cartridges (Thermo Scientific, USA) as per manufacturer's instructions. The purified antibody was analysed by SDS-PAGE and measured quantitatively by UV absorption. The immunoglobulin subclass was determined using a mouse monoclonal antibody IsoQuick typing kit (Sigma-Aldrich, St. Louis, MO) as per manufacturer's instructions.
Antigen specificity of MAbs by ELISA. The specificity of produced monoclonal antibodies was tested by IgG ELISA against recombinant LigA (LK90) protein and heat killed antigens prepared from MACS 13 of different leptospiral serovars, and heat killed antigens from bacterial pathogens other than Leptospira. Two micrograms of each representative proteins per well were coated on a flat-bottom polystyrene microtiter plate at 4 °C overnight, using carbonate coating buffer (pH 9.6), followed by blocking with 4% non-fat dry milk. Purified monoclonal antibodies (1:100) in triplicate were added and incubated for 1 h at 37 °C. Bound IgG was detected using HRP-conjugated rabbit anti-mouse IgG (Sigma-Aldrich, St. Louis, MO) at a dilution of 1:8,000. Plates were developed with o-phenylenediamine (Sigma-Aldrich, St. Louis, MO). The reaction was stopped with the addition of 50 µl of 1 N H 2 SO 4 , and the optical density was measured at 490 nm (Bio-Rad, USA). Dot Blot ELISA. As we propose the final end product of the developed MAb is its utilization in dot-blot ELISA, the specificity of MAbs was tested against recombinant LigA (LK90) protein, heat killed antigens prepared from MACS 13 of different leptospiral serovars, and heat killed antigens from freshly isolated bacterial pathogens other than Leptospira. To assay the utilization of MAbs for antigen capture, one microgram of pretreated urine samples from patients and rodents were dotted individually onto nitrocellulose (NC) strips. Urine samples from healthy individuals and whole cell lysates of leptopsiral serovars were used as negative and positive controls respectively. The NC strips were air dried and blocked with 5% skim milk in PBS (pH 7.4), incubated at room temperature for 1 h and washed thrice (10 mins each) with 0.01 M PBS (pH 7.4). Then the NC strips were incubated with MAbs (5 µg/ml) for 1 h at room temperature. The NC strips were washed with PBST (pH 7.4) as described above and then incubated with HRP conjugated rabbit anti-mouse IgG (1:8,000) for 1 h at room temperature. After incubation the strips were washed (3 times, 10 mins each) and immersed in chromogenic substrate 4-chloro-1-napthol (Sigma-Aldrich, St. Louis, MO) and 30% H 2 O 2 solution for 5 min, washed with distilled water and air dried. The colour intensity of dots were analysed by Image J software.
Data analysis. Data were analyzed using Statistica software package version 6.0 (StatSoft, Tulsa, OK). Cut-off values were defined as the corresponding Mean + 2 SD from the sera of healthy controls. Sensitivity was defined as the percentage of the laboratory-confirmed cases of leptospirosis whose serum samples gave mean ODs greater than the relevant cutoff value. Specificity was calculated as the percentage of the control individuals whose samples gave mean ODs below the relevant cutoff value. The positive and negative predictive values (PPV and NPV, respectively) are the proportions of true positives and true negatives, respectively. The percent agreement between the results of the recombinant protein-based ELISA and the results of the MAT, and the corresponding kappa coefficients, were determined using Epi Info version 6.0 (Centers for Disease Control and Prevention, Atlanta, GA) 13 . Graphs were made using SigmaPlot version 11.0 (Systat Software, Inc.) and GraphPad Prism version 5.0 (GraphPad, San Diego, CA). ed.
Hybridoma production and isotyping of MAbs. A total of 2 × 10 8 spleen cells obtained from immunized mice with the highest titre in indirect ELISA (1:10240) were fused with 2 × 10 7 myeloma cells. The culture supernatants from growing hybridomas were screened for antibody production with homologous peptides by IgG ELISA. More than ten hybridomas were found growing in each well of a microtitre plate after fusion yielding approximately 952 and 1243 hybridomas for LK90 543 and LK90 1110 respectively. The primary screening identified 62 (6.5%) and 82 (6.6%) hybridomas positive for LK90 543 and LK90 1110 respectively. Stable hybridoma clones for LK90 543 and LK90 1110 were designated as P1C5, P1B1 and P4E8, P4D1, P4W2 respectively. The produced MAbs had different isotypes, 4 (P1B1, P1C5, P4D1 and P4E8) of IgG1 types and one (P4W2) of IgG2b type and all with kappa light chains.
Antigen specificity of MAbs. The specificity and reactivity of the MAbs against leptospiral serovars and recombinant LK90 protein were determined by Western blot analysis and ELISA. The overall results of IgG ELISA for specificity and reactivity is given in Fig. 2. The mean + 2 SD of the absorbance values for other bacterial antigens were defined as the cut off value. The cut-off values for P1C5, P1B1, P4E8, P4D1, and P4W2 were 0.12, 0.134, 0.145, 0.161, and 0.169 respectively. The clone P1B1 and P4W2 were found to be highly specific for leptospiral serovars and hence selected for further experiments. To further confirm the specificity of the hybridomas, heat killed antigens of different leptospiral serovars (MACS), E. coli, and the recombinant LK90 protein were separated on SDS-PAGE and probed with purified P1B1 and P4W2 MAbs. The produced MAbs did not show cross-reactivity with E. coli further confirming its specificity for leptospiral antigens (Fig. 3). The specificity of P1B1 and P4W2 to detect leptospiral antigens were found to be in the range of ~93-96%. In order to further validate the specificity of the MAbs to detect only leptospiral antigens, dot blot ELISA was performed utilizing heat killed leptospiral antigens (MACS), and antigens from other pathogenic bacteria. The dot blot ELISA show the produced MAbs to be highly specific to leptopsiral antigens with no detectable cross reactivity with other antigens (Fig. S1).
Dot-blot ELISA. Dot blot ELISA was developed with urine samples collected from human subjects and rodents. The optimal concentration of antigen that can be captured by MAbs was determined to be 1 μg (Fig. S2). Then we asked whether the developed MAbs can be employed in an antigen-capture dot blot format for diagnosis of leptospirosis. To test this, we first determined the concentration of MAbs that can detect letopsiral antigens (Fig. S3). The optimized concentration of MAbs were determined to be 5 μg and was used in dot blot ELISA. The specificity of the MAbs to capture leptospiral antigen in urine was determined using samples collected from experimentally infected BALB/c mice. The MAbs showed 100% specificity and sensitivity in capturing the leptospiral antigen (Fig. S4). Further we determined the diagnostic efficacy of the MAbs using human and rodent samples. The overall sensitivity and specificity of the Ag-capture dot-blot ELISA developed with MAbs are given in Table 1 and Fig. 4. The specificity of P1B1 and P4W2 to detect leptospiral antigens were found to be in the range of ~93-96%.

Calculation of Incremental-cost effectiveness ratio (ICER).
After establishing the importance of dot-blot ELISA in the early diagnosis of leptospirosis, we intended to evaluate the cost effectiveness of the developed strategy. We used decision tree model to determine the cos-effectiveness. The decision tree was made based on the sensitivity of the dot-blot ELISA. Incremental cost effectiveness ratio (ICER) was calculated on comparison with the gold standard MAT. The parameters considered were the probability of each consequence (sensitivity; true positive), calculated approximate cost of the consequence, and the Quality Adjusted life years (QALY). Note: most febrile illness and particularly leptospirosis progress over a 10-15-day period, during which the patients make various possible transitions. Hence a 15-day limit was applied because most patients became afebrile within this period. Thus the model estimated the QALY as the number of no-fever days associated with each strategy. Our primary comparison was between; Dot-blot ELISA: sensitivity of 89%, approximate cost of $2.2 per test, and diagnostic delay of 4 days; MAT: sensitivity of 50%, cost of $4.5 per test and approximately diagnostic delay of 10 days. The model yielded an Incremental Cost-Effectiveness Ratio (ICER) as $8.71/QALY (Fig. S5; and Table S5).

Discussion
Leptospirosis is a serious public health problem that is generally underdiagnosed. The misdiagnosis of the disease is highly fatal due to potential damage to multiple organs. The method most commonly used for the diagnosis of human and animal leptospirosis is detection of specific serum antibody by MAT, and ELISA. These serological methods depend on the presence of circulating antibody and have variable sensitivities and specificities. And moreover, antibodies are detected in the blood approximately 4-7 days after the onset of the symptoms. The development of an early, sensitive and reliable diagnostic format would improve quality of patients' life. A large array of advanced diagnostic formats including qPCR to detect circulating antigens in blood have been developed 16 . However these diagnostic techniques suffer from several drawbacks like, available only in well-equipped laboratories, needs specially trained persons for performing the assay and not all clinical laboratories achieve same level of diagnostic excellence. Therefore, there is an increased search for a simpler and rapid technique that can be performed in any diagnostic laboratories with minimum laboratory facilities including a table top centrifuge to prepare antigenic samples, and a refrigerator to store specimens. In the acute phase of illness, the leptospires disseminate to visceral organs through blood stream of the infected persons 17 and are excreted in urine from day 6 post-infection onward 18 . Therefore, the detection of leptospiral antigens using precise antibodies will be an alternative strategy for the diagnosis of leptospirosis during the early stages of infection.   Monoclonal antibody based antigen detection methods have been increasingly used in detection of many bacterial and viral infections 19 . Nguyen et al., 20 used MAbs for the detection of typhoid during the early phase of illness. Initially the MAb has been produced for whole cell lysate of leptospires which showed specificity to its serovar and failed to detect others 4,21 . The monoclonal antibody 1H6 was produced against leptospiral lipopolysaccharide 22 . The 1H6 MAb based immunochromatography showed a sensitivity and specificity of 89 and 87% respectively. On the other hand, the recombinant proteins which are surface exposed and conserved have also been used to develop monoclonal antibodies with remarkable diagnostic efficiencies 23 . Leptospiral immunoglobulin-like (Lig) proteins are in-vivo expressed surface protein that has gained attention as diagnostic and vaccine candidate. Detection of leptospirosis using recombinant C-terminal LigA described achievable specificity values of 100% and 98% for IgG and IgM ELISAs, respectively 24 . Serodiagnosis of leptospirosis using chemically synthesised peptides of LigA showed higher sensitivity and specificity of 97.9% and 99.1% respectively 8 . Therefore, the chemically synthesised peptides of LigA can be considered as effective antigens for production of monoclonal antibody.
In the present study, we have computationally identified immunogenic B-cell epitopes of LK90 (LigA-90kDa), immunized mice with synthetic peptides and developed monoclonal antibodies. Two positive stable clones P1B1 (LK90 543 ) and P4W2 (LK90 1110 ) were selected and used in dot-blot ELISA to detect antigens. Peptide based monoclonal antibody offers high specificity in terms of monovalent antigenicity and Leptospira specific diagnosis. We have explored the applicability of the MAb based dot-blot ELISA for the detection of leptospiral antigen in urine samples. The dot-blot ELISA developed with these MAbs were found to be highly specific for conclusive diagnosis of acute leptospirosis during outbreak situations.
For specificity assays, the MAbs obtained were tested against several leptospiral serovars and other bacterial pathogens. The MAbs were able to detect the native proteins in all leptospiral serovars with a specificity of 100% and no cross reactivity with non-leptospiral bacteria as evidenced by Western blotting and IgG ELISA. A major downside of the present study is that antigenic preparations from other spirochetes including Treponema/Borrelia were not included. Considering the exertion and our expertise in the laboratory cultivation of Treponema/Borrelia the antigens has been discounted. On the other hand, the efficiency of our developed MAbs was validated by incorporating clinical cases confirmed for other spirochetal diseases (Syphilis and Lyme). Additionally, pondering the use of paired sera to demonstrate the seroconversion or four-fold rise in titre for the laboratory confirmation of leptospirosis, the cross-reactivity of our MAbs with other spirochetes was debarred. The present strategies of developing MAbs have opened new avenues for the development of MAbs for other spirochetal antigens.
Further the dot-blot ELISA was applied to experimentally infected-and noninfected-mice urine samples. The culture method was used as a gold standard comparison for the calculation of sensitivity and specificity 25 . The sensitivity and specificity of the MAb based dot-blot ELISA were found to be 100%. Such increased sensitivity could possibly be due to high concentrations of leptospires in the infected mice urine samples. According to Monahan et al., 9 , infected rats could excrete high concentrations of Leptospira (10 7 cells/ml) after 3 weeks of infection.
Urine samples from 32 patients with laboratory confirmed leptospirosis, 28 healthy humans, and 33 field rats were tested using the MAb based dot-blot ELISA. MAT was performed and used as gold standard for diagnosis of leptospirosis. The specificity was between 93 and 96% and sensitivity between 77 and 89%. The decrease in sensitivity might be due to nonspecific reactions with unknown substances in the urine. Therefore, pre-treatment of urine samples will be necessary to eliminate these substances. But there were no sensitivity issues when experimentally infected mice urine samples were used for dot-blot ELISA. This could possibly be due to the higher concentrations of leptopsires over the non-specific substances. However, further study is necessary in order to create simpler and more-cost-effective methods of pre-treating urine samples and to determine the range of leptospiral antigen concentration that could be detected without compromising the assay sensitivity.
The concentration of Leptospira that is usually found in urine from dogs ranges from 10 1 to 10 6 cells/ml 26 . The concentration of leptospires varies among animals with variable assay sensitivity. This is a limitation of our study and further improvements are needed for the wide applicability of the produced MAbs.
In conclusion, the MAb based antigen detecting dot-blot ELISA from urine sample has been developed for the diagnosis of acute leptospirosis. Since the test doesn't need any special equipment including PCR, western blot apparatus, dark field microscopy maintaining of live leptospires and invasive procedure for the collection of specimen from patients, it is considered as an effective alternative strategy for diagnosis of acute leptospirosis in primary health care centres. And moreover dot-blot ELISA is simple, rapid can be performed in less than 8 hours (including incubation with MAb for 1 h, followed by washing for 30 mins, incubation of secondary antibody for 2 h, washing for 30 mins, and development, visualization and interpretation in 30 mins), inexpensive and affordable in developing countries and area where laboratory facilities are limited. Conclusively, the MAbs obtained were able to detect the native proteins in several leptospiral serovars as evidenced by Western blotting and IgG ELISA. The dot-blot ELISA developed with these MAbs is highly specific for conclusive diagnosis of acute leptospirosis during outbreak situations.