TY - JOUR
T1 - Monoclonal antibodies to intracellular stages of Cryptosporidium parvum define life cycle progression in vitro
AU - Wilke, Georgia
AU - Ravindran, Soumya
AU - Funkhouser-Jones, Lisa
AU - Barks, Jennifer
AU - Wang, Qiuling
AU - VanDussen, Kelli L.
AU - Stappenbeck, Thaddeus S.
AU - Kuhlenschmidt, Theresa B.
AU - Kuhlenschmidt, Mark S.
AU - Sibley, L. David
N1 - Funding Information:
This work was supported by Grand Challenges grants from the Bill and Melinda Gates Foundation (OPP1098828, OPP1139330). K. L. VanDussen was supported by a K01 grant from the NIH (DK109081). G. Wilke and L. Funkhouser-Jones were partially supported by an Institutional Training grant to Washington University (AI007172). The monoclonal lines derived in this paper were isolated from hybridomas created by Misha Hart and Paul Schjetnan under the guidance of Kathy Sheehan in the Washington University Hybridoma Facility, which is supported by the Facility of Rheumatic Diseases Core Center (NIH P30AR048335). We thank Wandy Beatty of the Microbiology Imaging Facility for electron microscopy processing. We thank Michael Arrowood and Honorine Ward for reagents and helpful advice on antibody staining. The life cycle stage cartoons used in this publication were created by Laura Kyro. All of the antibodies described here are available for research use, and interested parties should contact the corresponding author to arrange transfer of materials. G.W. and K.L.V.D. optimized the mIEC culture system for C. parvum infection. G.W., S.R., Q.W., and J.B. generated and characterized the hybridomas. S.R. subcloned the hybridomas. G.W., S.R., and L.F.-J. performed immunofluorescence staining analysis, T.B.K., M.S.K., and K.L.V.D. provided key biological reagents and advice, T.S.S. and L.D.S. provided advice and supervision, and G.W. and L.D.S. wrote the paper with input from all authors
Funding Information:
This work was supported by Grand Challenges grants from the Bill and Melinda Gates Foundation (OPP1098828, OPP1139330). K. L. VanDussen was supported by a K01 grant from the NIH (DK109081). G. Wilke and L. Funkhouser-Jones were partially supported by an Institutional Training grant to Washington University (AI007172). The monoclonal lines derived in this paper were isolated from hybridomas created by Misha Hart and Paul Schjetnan under the guidance of Kathy Sheehan in the Washington University Hybridoma Facility, which is supported by the Facility of Rheumatic Diseases Core Center (NIH P30AR048335).
Publisher Copyright:
© 2018 Wilke et al.
PY - 2018/5/1
Y1 - 2018/5/1
N2 - Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti-Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology.
AB - Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti-Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology.
KW - Cryptosporidium
KW - Cytoskeleton
KW - Intracellular parasites
KW - Membrane proteins
KW - Monoclonal antibodies
KW - Sexual development
UR - http://www.scopus.com/inward/record.url?scp=85048222593&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85048222593&partnerID=8YFLogxK
U2 - 10.1128/mSphere.00124-18
DO - 10.1128/mSphere.00124-18
M3 - Article
C2 - 29848759
AN - SCOPUS:85048222593
SN - 2379-5042
VL - 3
JO - mSphere
JF - mSphere
IS - 3
M1 - e00124-18
ER -