A Protein-Conjugate Approach to Develop a Monoclonal
Antibody-Based Antigen Detection Test for the
Diagnosis of Human Brucellosis
Kailash P. Patra1, Mayuko Saito1, Vidya L. Atluri2, Hortensia G. Rolán2, Briana Young2, Tobias Kerrinnes2,
Henk Smits3, Jessica N. Ricaldi4, Eduardo Gotuzzo4, Robert H. Gilman5,6, Renee M. Tsolis2*,
Joseph M. Vinetz1,4,6*
1 Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California, United States of America, 2 Department of Medical
Microbiology, University of California Davis, Davis, California, United States of America, 3 Department of Biomedical Research, Royal Tropical Institute, Amsterdam, the
Netherlands, 4 Institute of Tropical Medicine Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru, 5 Department of International Health, Johns
Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America, 6 Laboratory of Research and Development, and Department of Cellular and
Molecular Sciences, Faculty of Sciences, Universidad Peruana Cayetano Heredia, Lima, Peru
Abstract
Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen.
Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence
of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella
melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic
protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained
that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that
detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian
subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis
lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and
negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive
serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-
reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for
Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-
independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved
rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.
Citation: Patra KP, Saito M, Atluri VL, Rolán HG, Young B, et al. (2014) A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection
Test for the Diagnosis of Human Brucellosis. PLoS Negl Trop Dis 8(6): e2926. doi:10.1371/journal.pntd.0002926
Editor: Pamela L. C. Small, University of Tennessee, United States of America
Received September 29, 2013; Accepted April 20, 2014; Published June 5, 2014
Copyright: ß 2014 Patra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by United States Public Health Service grants 1U01AI075420, K24AI068903, and D43TW007120. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: (RMT); (JMV)
Introduction the use of 2-mercaptoethanol to distinguish IgG from IgM
antibodies when determining the presence of active infection
Human brucellosis is most commonly caused by two species of requiring antibiotic therapy; newer data obtained using genome-
the genus Brucella, typically B. abortus from cattle and B. melitensis level screens suggest the potential utility of recombinant B.
from goats and sheep. The definitive diagnosis of brucellosis rests melitensis proteins for characterization of human infection [22–24].
upon demonstration of the causative bacterium in a suspected Sometimes, when prozone or other interfering immune phenom-
patient’s body fluid, typically by culture isolation [1,2]. While ena occur where clinical brucellosis may be associated with non-
detection of Brucella nucleic acids [3–13] or antigens [14] would be agglutinating antibodies, the Coomb’s indirect antibody test or the
expected to be diagnostic for new cases of brucellosis, DNA has BrucellaCapt assay can detect anti-Brucella antibodies [19,25–32].
been reported to persist in blood after successful treatment of ELISA to detect IgM or IgG antibodies that react with B. abortus
solidly diagnosed cases [15,16]. Therefore PCR amplification- lysates are not recommended for diagnosis because of limited
based tests are not useful to confirm brucellosis relapse [15,16]. specificity, but a competitive ELISA to detect smooth Brucella LPS
Because culture is technically challenging and hazardous in many [33] and a rapid antibody-detecting test such as the lipopolysac-
clinical laboratories, brucellosis is most commonly diagnosed using charide (LPS)-based lateral flow assay has favorable performance
serological methods that use fixed, whole Brucella abortus as antigen characteristics [19,25–32]. Nonetheless, ELISA tests based on
[17–21]. Such methods include the Rose Bengal, slide agglutina- whole cell B. abortus lysates may suffer from false positive results.
tion, and tube agglutination tests, sometimes accompanied with False positive serological results may be also found with other
PLOS Neglected Tropical Diseases | www.plosntds.org 1 June 2014 | Volume 8 | Issue 6 | e2926
, Antigen Capture to Diagnose Brucellosis
Author Summary St. Louis, MO), and the hot phenol-water treatment was repeated.
B. melitensis LPS fractions obtained from both upper phenol
Brucellosis is a OneHealth disease reflecting the risk for saturated aqueous layer (aqueous phase) and lower water saturated
human infection by interaction with and relation to phenol layer (phenol phase) were pooled. Purified LPS was
affected animal populations. The disease is often difficult analyzed on a 4–12% gradient Tris-Glycine sodium dodecyl
to diagnose because of lack of precise or accessible sulfate (SDS) polyacrylamide gel (Invitrogen Corp., Carlsbad, CA)
diagnostic reagents, and because culture is complex, under reducing conditions. The presence of LPS in the gels was
hazardous and relatively insensitive. Brucellosis dispropor- detected with a periodic acid silver stain [41] and protein with
tionately affects the poor and dispossessed with human Coomassie blue stain (BioRad, Hercules, CA). B. melitensis LPS was
and animal burdens of disease in the Middle East, North quantified using a colorimetric assay to measure 2-keto-3-
Africa, Mongolia and other regions that are simply
deoxyoctonate (KDO) concentration [42]. E. coli 055: B5 LPS
unknown. The diagnosis of brucellosis most often rests
(Sigma Chemicals) was used as standard. We also extracted LPS
on serological tests—antibody detection—based on ag-
glutination of fixed Brucella abortus. We have developed from other Brucella species (B. abortus, B. suis, B. canis, B. ovis),
the basis for developing a new test based on the detection Yersinia enterocolitica 09 and B. melitensis manB mutant by following the
of the B. melitensis lipopolysaccharide, which provides earlier mentioned procedure.
rapid and definitive identification of the presence of the
organism in clinically obtainable body fluids. A new Keyhole limpet hemocyanin (KLH) conjugation to B.
approach—protein conjugation to the lipopolysaccharide melitensis LPS and monoclonal antibody development
antigen—was taken to enhance the affinity of the Three mg of purified B. melitensis LPS was used for conjugation
monoclonal antibodies that were generated for the test. with KLH (SoluLink, Inc., San Diego, CA). Briefly, periodate-
These reagents were tested in a mouse model of B. treated B. melitensis 16M LPS was linked to succinimidyl 4-
melitensis and in humans from the brucellosis-endemic
hydrazinonicotinate (SANH)-modified KLH. To estimate conju-
region of Peru, and provided the data for the basis of
gation efficiency, KLH-conjugated B. melitensis LPS was analyzed
further clinical development and clinical trials for the rapid,
point-of-care diagnosis of brucellosis that will also provide on 10% Bis-Tris SDS PAGE gels (Invitrogen Corp., Carlsbad,
new tools for assessing the global burden of disease. U.S.A) under reducing conditions and stained with periodic acid
silver that detects LPS.
Monoclonal antibodies were raised against KLH-conjugated B.
pathogenic bacteria because of cross-reaction with E. coli melitensis LPS using standard methods [43,44]. The primary screen
O157:H7, Francisella tularensis, Yersinia enterocolica and Salmonella for hybridoma supernatants was an ELISA using wells coated with
typhi (at low dilutions) can confound serological diagnosis but B. melitensis LPS. Positive hybridoma supernatants were isotyped
diseases caused by these agents are rarely confused with brucellosis and IgG isotypes chosen for further study (Monoclonal Antibody
[34–39]. Nonetheless, serological diagnosis provides only an Isotyping Kit, Pierce, Rockford, IL).
indirect measure of infection.
The present investigation aimed to develop new monoclonal
LPS immunoblot analysis
antibodies against the immunodominant LPS of B. melitensis,
To determine the reactivity of monoclonal antibodies to B.
towards the development of new tools for the direct detection of
melitensis LPS, an immunoblotting analysis was performed as
Brucella LPS antigen for diagnostic purposes. We adopted a new
described previously [45]. B. melitensis LPS was electrophoresed on
approach to enhance the affinity of IgG antibodies for the LPS
a 4–12% gradient Tris-Glycine SDS polyacrylamide gel and
antigen by coupling purified B. melitensis LPS to keyhole limpet
transferred to a nitrocellulose membrane using standard methods.
hemocyanin (KLH) prior to immunization and boosting, which
The blotted membrane was blocked (Superblock, Thermo Fisher
would be predicted to induce T cell-dependent affinity maturation
Scientific, Lake Barrington, IL) and strips were incubated
of the anti-LPS antibody response by B cells. Supernatants from
individually in neat hybridoma supernatants for 2 h at room
hybridomas which screened positive for anti-B. melitensis LPS by
temperature. The strips were washed three times in TBS-Tween
ELISA were further characterized by Western blot and indirect
20, 0.05% (TBST) and incubated with 1:3000 dilution (1%
immunofluorescence microscopy. A capture ELISA using the
Superblock in TBST) of goat anti-mouse IgG + IgM phosphatase
purified monoclonal antibodies was tested for its ability to detect B.
labeled antibodies (KPL, Maryland, U.S.A) for 1 h and washed
melitensis LPS antigen in sera from experimentally infected mice
four times in TBST. The blots were incubated in substrate solution
and Peruvian patients diagnosed with brucellosis by blood culture.
(BCIP/NBT-1 Phosphatase Substrate, KPL) for the color devel-
opment.
Methods To evaluate the cross reactivity of monoclonals, LPS of other
Purification of Brucella melitensis lipopolysaccharide Brucella species (B. abortus 2308 and B. suis 1330) and mutant (B.
Brucella melitensis 16M was grown under aeration to stationary melitensis manB), E. coli 0157:H7 and Y. enterocolitica were
phase at 37uC in tryptic soy broth (TSB). Cells were recovered by electrophoresed, transferred and probed with hybridoma super-
centrifugation and approximately 10 g of pelleted cells were natants as described above. The dilution of the hybridoma
inactivated by autoclaving (121uC, 40 min). Cell pellets were used supernatant varies between from 1:50 to 1: 10,000) based on the
to isolate lipopolysaccharide (LPS) using the hot phenol-water signal intensity and it’s mentioned in the blot for each monoclonal
method [40]. All manipulations of live Brucella melitensis were antibodies.
performed under Biosafety Level 3 conditions approved by the
Select Agent program carried out at the University of California, Specificity of monoclonal antibodies against B. melitensis
Davis under conditions established and supervised by the United LPS by indirect immunofluorescence microscopy
States Department of Agriculture and the United States Centers ELISA-positive hybridoma supernatants were further screened
for Disease Control and Prevention. The purified LPSs were by immunofluorescence against acetone-fixed B. melitensis 16 M,
treated with RNase, DNase and proteinase-K (Sigma Chemicals, E. coli 0157:H7 strain EDL933 and E. coli strain DH10B
PLOS Neglected Tropical Diseases | www.plosntds.org 2 June 2014 | Volume 8 | Issue 6 | e2926
Antibody-Based Antigen Detection Test for the
Diagnosis of Human Brucellosis
Kailash P. Patra1, Mayuko Saito1, Vidya L. Atluri2, Hortensia G. Rolán2, Briana Young2, Tobias Kerrinnes2,
Henk Smits3, Jessica N. Ricaldi4, Eduardo Gotuzzo4, Robert H. Gilman5,6, Renee M. Tsolis2*,
Joseph M. Vinetz1,4,6*
1 Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California, United States of America, 2 Department of Medical
Microbiology, University of California Davis, Davis, California, United States of America, 3 Department of Biomedical Research, Royal Tropical Institute, Amsterdam, the
Netherlands, 4 Institute of Tropical Medicine Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru, 5 Department of International Health, Johns
Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America, 6 Laboratory of Research and Development, and Department of Cellular and
Molecular Sciences, Faculty of Sciences, Universidad Peruana Cayetano Heredia, Lima, Peru
Abstract
Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen.
Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence
of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella
melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic
protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained
that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that
detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian
subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis
lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and
negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive
serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-
reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for
Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-
independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved
rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.
Citation: Patra KP, Saito M, Atluri VL, Rolán HG, Young B, et al. (2014) A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection
Test for the Diagnosis of Human Brucellosis. PLoS Negl Trop Dis 8(6): e2926. doi:10.1371/journal.pntd.0002926
Editor: Pamela L. C. Small, University of Tennessee, United States of America
Received September 29, 2013; Accepted April 20, 2014; Published June 5, 2014
Copyright: ß 2014 Patra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by United States Public Health Service grants 1U01AI075420, K24AI068903, and D43TW007120. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: (RMT); (JMV)
Introduction the use of 2-mercaptoethanol to distinguish IgG from IgM
antibodies when determining the presence of active infection
Human brucellosis is most commonly caused by two species of requiring antibiotic therapy; newer data obtained using genome-
the genus Brucella, typically B. abortus from cattle and B. melitensis level screens suggest the potential utility of recombinant B.
from goats and sheep. The definitive diagnosis of brucellosis rests melitensis proteins for characterization of human infection [22–24].
upon demonstration of the causative bacterium in a suspected Sometimes, when prozone or other interfering immune phenom-
patient’s body fluid, typically by culture isolation [1,2]. While ena occur where clinical brucellosis may be associated with non-
detection of Brucella nucleic acids [3–13] or antigens [14] would be agglutinating antibodies, the Coomb’s indirect antibody test or the
expected to be diagnostic for new cases of brucellosis, DNA has BrucellaCapt assay can detect anti-Brucella antibodies [19,25–32].
been reported to persist in blood after successful treatment of ELISA to detect IgM or IgG antibodies that react with B. abortus
solidly diagnosed cases [15,16]. Therefore PCR amplification- lysates are not recommended for diagnosis because of limited
based tests are not useful to confirm brucellosis relapse [15,16]. specificity, but a competitive ELISA to detect smooth Brucella LPS
Because culture is technically challenging and hazardous in many [33] and a rapid antibody-detecting test such as the lipopolysac-
clinical laboratories, brucellosis is most commonly diagnosed using charide (LPS)-based lateral flow assay has favorable performance
serological methods that use fixed, whole Brucella abortus as antigen characteristics [19,25–32]. Nonetheless, ELISA tests based on
[17–21]. Such methods include the Rose Bengal, slide agglutina- whole cell B. abortus lysates may suffer from false positive results.
tion, and tube agglutination tests, sometimes accompanied with False positive serological results may be also found with other
PLOS Neglected Tropical Diseases | www.plosntds.org 1 June 2014 | Volume 8 | Issue 6 | e2926
, Antigen Capture to Diagnose Brucellosis
Author Summary St. Louis, MO), and the hot phenol-water treatment was repeated.
B. melitensis LPS fractions obtained from both upper phenol
Brucellosis is a OneHealth disease reflecting the risk for saturated aqueous layer (aqueous phase) and lower water saturated
human infection by interaction with and relation to phenol layer (phenol phase) were pooled. Purified LPS was
affected animal populations. The disease is often difficult analyzed on a 4–12% gradient Tris-Glycine sodium dodecyl
to diagnose because of lack of precise or accessible sulfate (SDS) polyacrylamide gel (Invitrogen Corp., Carlsbad, CA)
diagnostic reagents, and because culture is complex, under reducing conditions. The presence of LPS in the gels was
hazardous and relatively insensitive. Brucellosis dispropor- detected with a periodic acid silver stain [41] and protein with
tionately affects the poor and dispossessed with human Coomassie blue stain (BioRad, Hercules, CA). B. melitensis LPS was
and animal burdens of disease in the Middle East, North quantified using a colorimetric assay to measure 2-keto-3-
Africa, Mongolia and other regions that are simply
deoxyoctonate (KDO) concentration [42]. E. coli 055: B5 LPS
unknown. The diagnosis of brucellosis most often rests
(Sigma Chemicals) was used as standard. We also extracted LPS
on serological tests—antibody detection—based on ag-
glutination of fixed Brucella abortus. We have developed from other Brucella species (B. abortus, B. suis, B. canis, B. ovis),
the basis for developing a new test based on the detection Yersinia enterocolitica 09 and B. melitensis manB mutant by following the
of the B. melitensis lipopolysaccharide, which provides earlier mentioned procedure.
rapid and definitive identification of the presence of the
organism in clinically obtainable body fluids. A new Keyhole limpet hemocyanin (KLH) conjugation to B.
approach—protein conjugation to the lipopolysaccharide melitensis LPS and monoclonal antibody development
antigen—was taken to enhance the affinity of the Three mg of purified B. melitensis LPS was used for conjugation
monoclonal antibodies that were generated for the test. with KLH (SoluLink, Inc., San Diego, CA). Briefly, periodate-
These reagents were tested in a mouse model of B. treated B. melitensis 16M LPS was linked to succinimidyl 4-
melitensis and in humans from the brucellosis-endemic
hydrazinonicotinate (SANH)-modified KLH. To estimate conju-
region of Peru, and provided the data for the basis of
gation efficiency, KLH-conjugated B. melitensis LPS was analyzed
further clinical development and clinical trials for the rapid,
point-of-care diagnosis of brucellosis that will also provide on 10% Bis-Tris SDS PAGE gels (Invitrogen Corp., Carlsbad,
new tools for assessing the global burden of disease. U.S.A) under reducing conditions and stained with periodic acid
silver that detects LPS.
Monoclonal antibodies were raised against KLH-conjugated B.
pathogenic bacteria because of cross-reaction with E. coli melitensis LPS using standard methods [43,44]. The primary screen
O157:H7, Francisella tularensis, Yersinia enterocolica and Salmonella for hybridoma supernatants was an ELISA using wells coated with
typhi (at low dilutions) can confound serological diagnosis but B. melitensis LPS. Positive hybridoma supernatants were isotyped
diseases caused by these agents are rarely confused with brucellosis and IgG isotypes chosen for further study (Monoclonal Antibody
[34–39]. Nonetheless, serological diagnosis provides only an Isotyping Kit, Pierce, Rockford, IL).
indirect measure of infection.
The present investigation aimed to develop new monoclonal
LPS immunoblot analysis
antibodies against the immunodominant LPS of B. melitensis,
To determine the reactivity of monoclonal antibodies to B.
towards the development of new tools for the direct detection of
melitensis LPS, an immunoblotting analysis was performed as
Brucella LPS antigen for diagnostic purposes. We adopted a new
described previously [45]. B. melitensis LPS was electrophoresed on
approach to enhance the affinity of IgG antibodies for the LPS
a 4–12% gradient Tris-Glycine SDS polyacrylamide gel and
antigen by coupling purified B. melitensis LPS to keyhole limpet
transferred to a nitrocellulose membrane using standard methods.
hemocyanin (KLH) prior to immunization and boosting, which
The blotted membrane was blocked (Superblock, Thermo Fisher
would be predicted to induce T cell-dependent affinity maturation
Scientific, Lake Barrington, IL) and strips were incubated
of the anti-LPS antibody response by B cells. Supernatants from
individually in neat hybridoma supernatants for 2 h at room
hybridomas which screened positive for anti-B. melitensis LPS by
temperature. The strips were washed three times in TBS-Tween
ELISA were further characterized by Western blot and indirect
20, 0.05% (TBST) and incubated with 1:3000 dilution (1%
immunofluorescence microscopy. A capture ELISA using the
Superblock in TBST) of goat anti-mouse IgG + IgM phosphatase
purified monoclonal antibodies was tested for its ability to detect B.
labeled antibodies (KPL, Maryland, U.S.A) for 1 h and washed
melitensis LPS antigen in sera from experimentally infected mice
four times in TBST. The blots were incubated in substrate solution
and Peruvian patients diagnosed with brucellosis by blood culture.
(BCIP/NBT-1 Phosphatase Substrate, KPL) for the color devel-
opment.
Methods To evaluate the cross reactivity of monoclonals, LPS of other
Purification of Brucella melitensis lipopolysaccharide Brucella species (B. abortus 2308 and B. suis 1330) and mutant (B.
Brucella melitensis 16M was grown under aeration to stationary melitensis manB), E. coli 0157:H7 and Y. enterocolitica were
phase at 37uC in tryptic soy broth (TSB). Cells were recovered by electrophoresed, transferred and probed with hybridoma super-
centrifugation and approximately 10 g of pelleted cells were natants as described above. The dilution of the hybridoma
inactivated by autoclaving (121uC, 40 min). Cell pellets were used supernatant varies between from 1:50 to 1: 10,000) based on the
to isolate lipopolysaccharide (LPS) using the hot phenol-water signal intensity and it’s mentioned in the blot for each monoclonal
method [40]. All manipulations of live Brucella melitensis were antibodies.
performed under Biosafety Level 3 conditions approved by the
Select Agent program carried out at the University of California, Specificity of monoclonal antibodies against B. melitensis
Davis under conditions established and supervised by the United LPS by indirect immunofluorescence microscopy
States Department of Agriculture and the United States Centers ELISA-positive hybridoma supernatants were further screened
for Disease Control and Prevention. The purified LPSs were by immunofluorescence against acetone-fixed B. melitensis 16 M,
treated with RNase, DNase and proteinase-K (Sigma Chemicals, E. coli 0157:H7 strain EDL933 and E. coli strain DH10B
PLOS Neglected Tropical Diseases | www.plosntds.org 2 June 2014 | Volume 8 | Issue 6 | e2926
