Testing different antigen capture ELISA formats for detection of Leptospira spp. in human blood serum

Transactions of the Royal Society of Tropical Medicine and Hygiene 104 (2010) 259–264

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Testing different antigen capture ELISA formats for detection of Leptospira spp. in human blood serum Flávia A. Vasconcellos, Mariana L. Coutinho, Éverton F. da Silva, Cláudia P.H. Fernandes, Leonardo G. Monte, Núbia Seyffert, Odir Antônio Dellagostin, José Antonio G. Aleixo ∗ Centro de Biotecnologia, Universidade Federal de Pelotas, Cx. Postal 354, 96010-900, Pelotas, RS, Brazil

a r t i c l e

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Article history: Received 4 May 2009 Received in revised form 9 October 2009 Accepted 9 October 2009 Available online 25 November 2009 Keywords: Leptospirosis Leptospira Monoclonal antibodies Biotin Streptavidin Immunoglobulin Y

a b s t r a c t Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira. The illness is characterized by an acute bacteremic phase followed by an immune phase, in which specific antibodies are found in blood and leptospires are eliminated in urine. Novel diagnostic strategies for use in the acute phase of leptospirosis are needed since clinical manifestations in this phase mimic other feverish tropical diseases. In the present study, mAbs and polyclonal IgY were used in the standardization of three different antigen capture ELISA formats for direct detection of leptospires in human blood during the acute phase of the disease. Detection limit of leptospires in experimentally contaminated human sera ranged from 105 to 107 cells ml−1 in the different formats. The ELISA format with the best performance was able to detect 105 leptospires ml−1 in human sera using a mAb against LipL32, the major outer membrane protein of pathogenic leptospires, as capture antibody, and a biotinylated polyclonal IgY against a pathogenic serovar of L. interrogans Icterohamorrhagiae as detection antibody. By increasing the degree of IgY biotinylation this detection limit could be improved to make the assay clinically useful. © 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

1. Introduction Leptospirosis is a zoonosis caused by pathogenic spirochetes of the genus Leptospira that are transmitted directly or indirectly from animals to humans.1 Recently, leptospirosis has emerged as a globally important infectious disease.2 Incidence rates of human leptospirosis are underestimated, and there have been no precise estimates of the global burden due to lack of awareness of the disease, and relatively inaccessible and insufficiently rapid diagnostics.2 Leptospirosis is a life-threatening clinical manifestation and early diagnosis in the acute phase of infection is needed for prompt initiation of antibiotic therapy.3

∗ Corresponding author. Tel.: +55 53 32757583; fax: +55 53 32757551. E-mail address: [email protected] (J.A.G. Aleixo).

Current reference techniques for laboratory diagnosis of leptospirosis include the culture isolation of leptospires from patients and the microscopic agglutination test (MAT).1 Isolation of leptospires from blood, urine and tissues of patients is the gold standard of laboratory diagnosis, but this technique is of no use for early confirmation of leptospirosis. Use of MAT as the standard for serologic confirmation is laborious and impractical for rapid diagnosis since it requires analysis of paired sera samples, and the sensitivity and specificity are affected by variations between reference laboratories.2 Considerable numbers of immunoassays for detecting leptospiral antibodies4–6 and antigens7–9 have been employed to identify acute and convalescent-phase illness. Although some of these assays have been more sensitive than MAT, only a few are manufactured on a scale large enough for use in public health laboratories and the health care system.

0035-9203/$ – see front matter © 2009 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2009.10.005

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Advances in recombinant DNA technology have provided a new approach for rapid and sensitive diagnosis of infectious diseases. PCR has been tested for early diagnosis of leptospirosis.10–12 However, this molecular technique requires trained technicians and sophisticated laboratory equipment, so the need remains for development of accessible, sensitive and specific assays that can detect the acute phase of disease. Recognition of specific epitopes on pathogen surfaces by mAbs contributes positively to the specificity of immunoassays, while their scanty expression and conformational variations of antigen molecules contribute negatively to the sensitivity. On the other hand, polyclonal antibodies recognize several epitopes resulting in a stronger assay signal but with less specificity.13 The expression of several outer membrane proteins (OMPs) identified as putative virulence factors, present only in pathogenic leptospires and recognized by patient convalescent sera, allowed the production of mAbs against LipL3214,15 and LigA and LigB,16 which may be used for the development of immunoassays either together or combined with polyclonal antibodies. Chicken IgY presents several advantages for use in immunoassays compared to mammal IgG. It is harvested from eggs so avoids animal bleeding, and does not crossreact with rheumatoid factors or human anti-mouse antibodies, does not activate the mammal complement system and has less unspecific background.17 Furthermore, previous studies demonstrated that chicken IgY has affinity and sensitivity similar to mammal IgG in most common assays.17,18 We used IgY as a novel strategy for developing antigen capture ELISA for the laboratory diagnosis of the acute phase of leptospirosis. Three ELISA formats were tested for direct detection of leptospires in experimentally contaminated human blood using IgY against whole leptospiral cells and mAbs against OMPs of pathogenic species. This study was carried out in Pelotas city, RS, Brazil, in 2007.

Antibodies specific for the OMPs LipL3214–15 , LigA and LigB16 for use in the antigen capture ELISA were selected from a panel of mAbs after additivity index (AI) determination using a modification of a method described for epitope mapping.19 Briefly, an indirect ELISA was performed by adding the mAbs in pairs and individually to microtiter plates containing 107 leptospires per well. The amount of bound antibody was tested by adding goat anti-mouse Ig-peroxidase conjugated antibodies. An AI for each pair of mAbs binding to leptospires fixed in plate wells was calculated by comparing its absorbance value with the absorbance results of each single mAb binding as described.14 Additivity index values close to zero suggest that the antibodies being tested interfere with the binding of each other, which results in no amplification of absorbance values.

2. Materials and methods

2.4. IgY production

2.1. Leptospira strains and antigen preparation

Twenty-two-week-old White Leghorn chickens were used to produce polyclonal IgY antibodies. First, two hens were immunized by intramuscular injection of 108 cells of heat-inactivated whole-cell L. interrogans Fiocruz L1-130 in 125 ␮l of PBS, emulsified with 125 ␮l of Freund’s complete adjuvant (Sigma-Aldrich, St Louis, MO, USA). Second, third and fourth booster injections were given at 15 day intervals with the same dosage of antigen emulsified in Freund’s

Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and L. biflexa serovar Patoc 1 were obtained from the Centro de Pesquisa Gonc¸alo Moniz (FIOCRUZ, Salvador, Brazil). Leptospires were cultivated in Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium (Difco Laboratories, Detroit, MI, USA) at 29 ◦ C. Cul-

ture growth was monitored by dark-field microscopy as described.3 Whole-cell antigens were used for polyclonal IgY antibody production, immunoblotting and antigen capture ELISA. Antigens were prepared with cells from 7day cultures harvested by centrifugation (15,000 × g for 30 min) at 4 ◦ C and washed twice in PBS (0.01 M, pH 7.2). The cells were resuspended in PBS, counted in a PetroffHauser counting chamber (Fisher Scientific, Pittsburgh, PA, USA), and the concentration adjusted to approximately 108 cells ml−1 . The cells were then killed (56 ◦ C, overnight) and stored at −20 ◦ C. 2.2. Serum samples A pool of serum samples from healthy individuals donated by Hemocentro Regional de Pelotas/RS was confirmed negative for leptospirosis by MAT as described.1 Samples from the sera pool were contaminated experimentally with heat-inactivated Fiocruz L1-130 and Patoc strains. 2.3. Monoclonal antibodies and additivity index

Table 1 ELISA formats used for detection of leptospires in experimentally contaminated human sera ELISA format A B C a

Anti-LipL32. Anti-LigA. c Anti-LigB. b

Capture antibody a

mAb 1D9 mAb 1D9 mAb 1B12b mAb 3D10c

Detection antibody

Conjugate

IgY IgY biotinylated

Horseradish peroxidase- rabbit Ig anti-IgY Streptavidin-peroxidase

mAb 1D9 biotinylated

Streptavidin-peroxidase

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incomplete adjuvant. Eggs were collected from 3 days after the last immunization up to 30 days and were stored at 4 ◦ C until use. Extraction and purification of IgY from groups of four eggs was carried out as described.20 Concentration of IgY in the purified preparations was measured by spectrophotometry at 280 nm.21

Table 2 Additivity index for pairs of mAbs against LigA and LigB in indirect ELISA, using heat-inactivated Leptospira interrogans Fiocruz L1-130 as antigenb

2.5. SDS-PAGE and immunoblotting analysis For immunoblotting, antigens from heat-inactivated suspensions containing 109 leptospires ml−1 were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with blotto (5% milk in PBS) for 1 h. The IgY was diluted (1:300) in PBS and incubated with the membrane for 1 h at room temperature. After washing, the membrane was incubated with a rabbit immunoglobulin anti-IgY chicken horseradish peroxidase conjugate (Sigma) diluted 1:2000 in PBS and bands were visualized after addition of the substrate/chromogen solution (H2 O2 /cloronaftol). 2.6. Biotinylation of antibodies IgY and mAb 1D9 (anti-LipL32) were labeled with biotin using the method previously described.22 Initially, 1 mg of IgY or 1D9 was dissolved in 1 ml of phosphate-buffered saline (PBS 0.15 M; pH 7.2) and clarified by centrifugation at 4000 × g for 10 min at 4 ◦ C. The supernatant was dialyzed against sodium bicarbonate (0.1 M; pH 8.2) for 16 h at 4 ◦ C, again clarified and mixed with 1 ml of a fresh preparation of N-hydroxysuccinimido-biotin in DMSO (1 mg ml−1 ). The mix was shaken gently at room temperature for 4 h and dialyzed against PBS for 16 h. Another clarification was carried out by centrifugation and the supernatant was stored at −20 ◦ C. The degree of antibody biotinylation (molecules of biotin per molecule of antibody) was established using the EZ Biotin Quantitation Kit (Pierce, Rockford, IL, USA). The two biotinylated antibodies were then titrated in indirect ELISA using heat-inactivated leptospires or rLipL32 as antigens. 2.7. Antigen capture ELISA Three antigen capture ELISA formats (Table 1) were standardized through checkerboard titrations with different concentrations of capture and detection antibodies and conjugates. Microplate wells (Polysorp, Nunc International, Rochester, NY, USA) were coated with capture antibody in carbonate-bicarbonate buffer (0.05 M, pH 9.8) for 16 h at 4 ◦ C, followed by blocking with 0.5% BSA in PBS. Heatinactivated Fiocruz L1-130 or Patoc strains (108 cells ml−1 ) were then added and incubated for 1 h at room temperature. Leptospiral cells, detection antibodies and conjugates were diluted in PBS containing 0.05% Tween 20 (PBST) and plate wells were washed with PBS-T three times between each incubation step, except for conjugate which was washed five times. Presence of the antigen-antibody complex was revealed with a substrate/chromogen solution containing ortophenylendiamine (OPD, 2 mg ml−1 ) and hydrogen peroxide (5%) in 0.2 M citrate buffer (pH 4.0). The enzyme reaction was allowed to occur in darkness for

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b

mAb (protein)

1B12 (LigA)

1C7 (LigA)

1B4 (LigA)

1D1 (LigA)

3D10 (LigB) 1G7 (LigB) 5F12 (LigB) 3E4 (LigB) 3F12 (LigB) 4C2 (LigB)

46b 25 31 20 30 17

28 27 17 21 2 13

36 32 29 27 27 31

20 18 12 11 6 28

Results are averages of three independent experiments.

15 min and color development was measured at 450 nm (OD) in an ELISA reader (Titertek Multiskan MCC/340 MK II; Titertek Instruments, Inc., Huntsville, AL, USA). Reagent volumes were 100 ␮l/well and incubation steps were at room temperature for 1 h. 2.8. ELISA detection limits Limits of detection for the three antigen capture ELISA formats were determined using heat-inactivated cultures of Fiocruz L1-130 and Patoc strains ten-fold diluted in PBS and in a pool of sera from healthy individuals. 3. Results 3.1. Additivity index The AI experiment was performed to find out if the reaction with heat-inactivated Leptospira in the indirect ELISA would be improved by using pairs of mAbs against different OMPs, thus suggesting that they could be used together in the antigen capture ELISA. The highest additivity was obtained with mAb pair 3D10 and 1B12 that react with LigB and LigA, respectively (Table 2). Using this pair of mAbs plus mAb 1D9 (LipL32) increased AI even further (data not shown). 3.2. IgY production The mean yield of IgY was 31.7 mg (average of three purifications). SDS-PAGE used to investigate purity of preparations revealed one band with 67 kDa and another with 25 kDa under reducing conditions, and one band of 167 kDa under non-reducing conditions, corresponding to the molecular mass of the heavy and light chains and of the whole IgY molecule, respectively (Supplementary Figure 1). Purified IgY reacted with the same intensity with antigens from pathogenic and saprophytic leptospires in an indirect ELISA (Supplementary Figure 2). The profile of antigen reactivity with IgY investigated by Western blot was also very similar for both leptospires (Supplementary Figure 3). 3.3. Antibody biotinylation The number of biotin molecules per antibody molecule was 0.87 and 2.81 for IgY and mAb 1D9, respectively. Titration of biotinylated antibodies in indirect ELISA with the corresponding antigens indicated that IgY could be diluted

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Figure 1. Limit of detection of Leptospira interrogans in antigen capture ELISA. (A) mAb anti-LipL32 as capture antibody and IgY as detection antibody. Capture antibody: 0.2 ␮g per well. Detection antibody: diluted 1:300. Human sera = L. interrogans Fiocruz L1-130 (108 –103 ) diluted in human sera; PBS = L. interrogans Fiocruz L1-130 (108 –103 ) diluted in PBS; absorbance of negative controls: PBS = L. biflexa Patoc 108 cells ml−1 diluted in PBS = 0.095; human sera = L. biflexa Patoc 108 cells ml−1 diluted in human sera = 0.108. (B) mAb anti-LipL32 as capture antibody and biotinylated IgY as detection antibody. Capture antibody: 0.2 ␮g per well. Detection antibody: diluted 1:8000. Human sera = L. interrogans Fiocruz L1-130 (108 –103 ) diluted in human sera; PBS = L. interrogans Fiocruz L1-130 (108 –103 ) diluted in PBS; absorbance of negative controls: PBS = L. biflexa strain Patoc 108 cells ml−1 diluted in PBS = 0.290; human sera = L. biflexa strain Patoc 108 cells ml−1 diluted in human sera = 0.485. (C) mAbs anti-LigA and anti-LigB as capture antibodies and mAb 1D9 biotinylated as detection antibody. Capture antibody: 0.2 ␮g per well. Detection antibody: diluted 1:2000. Human sera = L. interrogans Fiocruz L1-130 (108 –103 ) diluted in human sera; PBS = L. interrogans Fiocruz L1-130 (108 –103 ) diluted in PBS; absorbance of negative control: PBS = L. biflexa Patoc 108 cells ml−1 diluted in PBS = 0.084; human sera = L. biflexa Patoc 108 cells ml−1 diluted in human sera = 0.111. All results are averages of three independent experiments.

1:8000 and mAb 1D9 1:2000 for use in the antigen capture ELISA (data not shown). 3.4. Antigen capture ELISA Three different antigen capture ELISA formats were tested in an attempt to identify the one capable of detecting the lowest number of leptospires in the blood. Format A, which used mAb 1D9 (anti-LipL32) as capture antibody and IgY as detection antibody, was able to detect 106 and 107 leptospires ml−1 in PBS or human serum, respectively (Figure 1A). In format B, which also used mAb 1D9 as capture antibody but used IgY biotinylated as detection antibody, the detection limit was between 105 and 106 leptospires ml−1 either in PBS or human serum (Figure 1B). Format C, which used the mAbs 1B4 (anti-LigA) and 3D10 (anti-LigB) as capture antibodies and mAb 1D9 biotinylated as detection antibody, detected between 106 and 107 leptospires ml−1 in PBS or human serum (Figure 1C). 4. Discussion In our study, we tested three different formats (A, B and C) of antigen capture ELISA for detection of leptospires in human sera experimentally contaminated. Format A used chicken polyclonal IgY as detection antibody to take advantage of properties such as absence of cross-reactions with mammalian antibodies and better cost-effective production in comparison with mammals, as IgY accumulate in large amounts in the egg yolk and can easily be purified.21

The average yield of IgY (31.7 mg yolk−1 ) was similar to that found in other studies21 , and the process of purification used allowed an appropriate degree of purity. However, it was found by indirect ELISA and Western blot that the reactivity profile of IgY with Fiocruz L1-130 and Patoc strains was very similar. This result was expected since leptospires have several immunogenic structures on their surfaces, including LPS, which may generate cross-reacting antibodies.3 mAb 1D9 was used as capture antibody in format A because of its high affinity for LipL32, the major OMP in pathogenic Leptospira. This format of antigen capture ELISA allowed detection of about 106 leptospires ml−1 of serum, which is a level of detection not appropriate since the blood density of leptospires in the acute phase of disease has been estimated to be 103 –104 ml−1 .23 Format B used biotinylated IgY together with a streptavidin-peroxidase conjugate in an attempt to increase the amount of enzyme in the antigen-antibody complex, thus increasing the end product of the enzymatic reaction and the subsequent amplification of the ELISA signal. This strategy was already used to increase the sensitivity of a range of immunochemical and molecular assays 24,25 because biotin can be easily conjugated to proteins and nucleic acids, and then detected with labeled streptavidin molecules, which have four binding sites with high affinity for biotin.26 Although our biotinylation process incorporated 0.87 biotin molecules per IgY molecule, this antigen capture ELISA format amplified the enzymatic signal and improved the detection limit, being able to detect 105 leptospires ml−1 of serum. Using an IgY with a higher number

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of biotin molecules may improve this ELISA enough to make it able to reach the appropriate detection limit for use in the acute phase of leptospirosis. The availability of a panel of mAbs against rLigANI, rLigBNI and rLipL32, allowed the standardization of a third format of antigen capture ELISA, format C. The choice of mAbs was based on results of an additivity assay, which provided information on the interference of mAbs on each other’s reaction. Based on the additivity results, mAbs 1B12 (anti-rLigANI) and 3D10 (anti-rLigBNI) were used as capture antibodies and biotinylated mAb 1D9 (anti-rLipL32) as detection antibody in this format. Even though the biotinylation of mAb 1D9 has been more efficient than IgY the detection limit of this ELISA was 106 to 107 leptospires ml−1 of serum, the highest among the three formats standardized. This was probably due to the low amount of mAb 1D9 retained in the bacterial surface. It is known that sandwich ELISA systems using mAbs as detection antibody have high detection limits due to loss of the amplifying effect produced by polyclonal sera.27 An alternative for increasing of the sensitivity of this antigen capture-ELISA may be using as detection antibody a mixture of mAbs against different leptospiral antigens. The development of more sensitive tests for early laboratory diagnosis of leptospirosis has been based mainly on IgM immunoassays, using whole-cell extracts or recombinant proteins as antigens5,28 , or PCR using conserved genes as targets of amplification.11 However, IgM tests have low sensitivity (<50%) in the initial stage of disease, while PCR, a highly sensitive test, requires sophisticated laboratory equipment that makes it difficult to use in laboratories from endemic areas. In this study, three different antigen capture ELISA formats were standardized for detection of leptospires in human sera experimentally contaminated. Format B, that uses mAbs as capture antibodies and biotinylated IgY as detection antibodies, showed potential for further adjustment to increase its detection limit of leptospires to the level found in blood in the acute phase of disease. This could be done by testing different mAbs as capture antibody and using as detection antibody IgY with a higher degree of biotinylation in order to get a better amplification of the antigen-antibody reaction. Authors’ contributions: FAV, OAD and JAGA planned the study and designed the protocol; CPHF, LGM, MLC, NS conducted the laboratory work and collected and managed the data; FAV, ÉFS and JAGA carried out the analyses and interpretation of the data and prepared the first draft of the manuscript. FAV and JAGA revised the paper. All authors read and approved the final manuscript. FAV and JAGA are guarantors of the paper. Acknowledgements: The authors would like to thank Prof. Marcos Anciutti from Universidade Federal de Pelotas (CAVG/UFPel) for all technical assistance. Funding: This work was supported by CNPq (Grant 558309/2008-9). FAV, LGM and NS were supported by CAPES Foundation (Brazilian Government), MLC, ÉFS,

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OAD and JAGA were supported by CNPq (Brazilian Government). Conflicts of interest: None declared. Ethical approval: The use of subject sera for these experiments was approved by the Ethical Research Committee from Universidade Federal de Pelotas, Pelotas, RS, Brazil. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.trstmh. 2009.10.005. References 1. WHO. Human leptospirosis: guidance for diagnosis, surveillance and control. Geneva: World Health Organization; 2003. 2. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, et al. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003;3:757–71. 3. Faine S, Adler B, Bolin C, Perolat P, editors. Leptospira and Leptospirosis. 2nd ed. Melbourne: MedSci; 1999. 4. Bomfim MR, Ko A, Koury MC. Evaluation of the recombinant LipL32 in enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis. Vet Microbiol 2005;109:89–94. 5. McBride AJA, Pereira FP, Silva ED, Matos RB, Silva ED, Ferreira AGP, et al. Evaluation of the EIE-IgM-Leptospirose assay for the serodiagnosis of leptospirosis. Acta Trop 2007;102:206–11. 6. Natarajaseenivasan K, Vijayachari P, Sharma S, Sugunan AP, Selvin J, Sehgal SC. Serodiagnosis of severe leptospirosis: evaluation of ELISA based on the recombinant OmpL1 or LipL41 antigens of Leptospira interrogans serovar autumnalis. Ann Trop Med Parasitol 2008;102:699–708. 7. Adler B, Chappel RJ, Faine S. The sensitivities of different immunoassays for detecting leptospiral antigen. Zentralbl Bakteriol Mikrobiol Hyg A 1982;252:405–13. 8. Champagne MJ, Ciggins R, Fairbrother JM, Dubreuil D. Detection and characterization of leptospiral antigens using a biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot. Can J Vet Res 1991;55:239–45. 9. Yan K-T, Ellis WA, Montgomery JM, Taylor MJ, Mackie DP, McDowell SWJ. Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine. Res Vet Sci 1998;64:119–24. 10. Fernandes CPH, Seixas FK, Coutinho ML, Vasconcellos FA, Moreira AN, Conceic¸ão FR, et al. An immunomagnetic separation-PCR method for detection of pathogenic Leptospira in biological fluids. Hybridoma 2008;27:1–6. 11. Jouglard SDD, Simionatto S, Seixas FK, Nassi FL, Dellagostin OA. Nested polymerase chain reaction for detection of pathogenic leptospiras. Can J Microbiol 2006;52:747–52. 12. Mérien F, Amouriaux P, Perolat P, Baranton G, Saint Girons I. Polymerase chain reaction for detection of Leptospira spp. J Clin Microbiol 1992;30:2219–24. 13. Campbell AM. Monoclonal and immunosensor technology. Amsterdam: Elsevier; 1991. 14. Coutinho ML, Vasconcellos FA, Fernandes CPH, Seyffert N, Seixas FK, Haake DA, et al. Evaluation of the anti-LipL32 monoclonal antibodies potential for use in leptospirosis immunodiagnostic tests. J Immunoassay Immunochem 2007;28:279–88. 15. Lüdtke CB, Coutinho ML, Jouglard SDD, Moreira CN, Fernandes CPH, Brod CS, et al. Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira. Braz J Microbiol 2003;34:1–4. 16. Seyffert N, Fernandes CPH, Vasconcellos FA, Coutinho ML, Raposo JB, Monte LG, et al. Monoclonal antibodies against leptospiral immunoglobulin-like proteins: production and characterization 107th General Meeting of the American Society for Microbiology; 21–25 May 2007; Toronto, Canada. 17. Narat M. Production of antibodies in chickens. Food Technol Biotechnol 2003;41:259–67. 18. Schade R, Calzado EG, Sarmiento R, Chacana PA, Porankiewicz-Asplund J, Terzolo HR. Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use

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