Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis

Kitland Louie, Karen W. Sverlow, Bradd C. Barr, Mark L Anderson, Patricia A Conrad

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle. For the serodiagnosis of this parasitic disease, two immunodominant clones from a bovine Neospora λgt11 library were identified, characterized, and expressed as recombinant proteins for the development of an enzyme-linked immunosorbent assay (ELISA). These two clones, designated N54 and N57, were 29 and 20 kDa, respectively, when expressed as histidine fusion proteins from the pRSET expression vector. Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecule; masses of 97, 87, 77, 67, and 64 kDa. Antibodies to recombinant protein N57 recognized four primary bands with molecular masses of 34, 31, 30, and 28 kDa. When a defined 'gold standard' panel of bovine Sera from confirmed Neospora-positive and Neospora-negative cattle were characterized by immunoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet. Binding to the N57 quadruplet was more variable. The same gold standard panel was used to evaluate and compare an N54-based ELISA, an N57-based ELISA, and a whole-tachyzoite lysate based ELISA. The sensitivities and specificities were 95 and 96% (N54 ELISA), 82 and 93% (N57 ELISA), and 74 and 93% (lysate ELISA). Thus, compared to the whole-tachyzoite lysate-based ELISA, both recombinant-protein-based ELISAs had higher sensitivities and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neosporosis.

Original languageEnglish (US)
Pages (from-to)692-699
Number of pages8
JournalClinical and Diagnostic Laboratory Immunology
Volume4
Issue number6
StatePublished - 1997

Fingerprint

Neospora
Recombinant proteins
Immunosorbents
Cloning
Serologic Tests
Recombinant Proteins
Organism Cloning
Assays
Enzyme-Linked Immunosorbent Assay
Enzymes
Molecular mass
Clone Cells
Antibodies
Parasitic Diseases
Histidine
Nervous System Diseases
Serum
Immunoblotting
Libraries
Proteins

ASJC Scopus subject areas

  • Microbiology (medical)
  • Immunology and Allergy
  • Clinical Biochemistry
  • Immunology

Cite this

Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis. / Louie, Kitland; Sverlow, Karen W.; Barr, Bradd C.; Anderson, Mark L; Conrad, Patricia A.

In: Clinical and Diagnostic Laboratory Immunology, Vol. 4, No. 6, 1997, p. 692-699.

Research output: Contribution to journalArticle

@article{35c764f0dbb54907b923cb0f8c7e1222,
title = "Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis",
abstract = "Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle. For the serodiagnosis of this parasitic disease, two immunodominant clones from a bovine Neospora λgt11 library were identified, characterized, and expressed as recombinant proteins for the development of an enzyme-linked immunosorbent assay (ELISA). These two clones, designated N54 and N57, were 29 and 20 kDa, respectively, when expressed as histidine fusion proteins from the pRSET expression vector. Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecule; masses of 97, 87, 77, 67, and 64 kDa. Antibodies to recombinant protein N57 recognized four primary bands with molecular masses of 34, 31, 30, and 28 kDa. When a defined 'gold standard' panel of bovine Sera from confirmed Neospora-positive and Neospora-negative cattle were characterized by immunoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet. Binding to the N57 quadruplet was more variable. The same gold standard panel was used to evaluate and compare an N54-based ELISA, an N57-based ELISA, and a whole-tachyzoite lysate based ELISA. The sensitivities and specificities were 95 and 96{\%} (N54 ELISA), 82 and 93{\%} (N57 ELISA), and 74 and 93{\%} (lysate ELISA). Thus, compared to the whole-tachyzoite lysate-based ELISA, both recombinant-protein-based ELISAs had higher sensitivities and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neosporosis.",
author = "Kitland Louie and Sverlow, {Karen W.} and Barr, {Bradd C.} and Anderson, {Mark L} and Conrad, {Patricia A}",
year = "1997",
language = "English (US)",
volume = "4",
pages = "692--699",
journal = "Clinical and Vaccine Immunology",
issn = "1556-6811",
publisher = "American Society for Microbiology",
number = "6",

}

TY - JOUR

T1 - Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis

AU - Louie, Kitland

AU - Sverlow, Karen W.

AU - Barr, Bradd C.

AU - Anderson, Mark L

AU - Conrad, Patricia A

PY - 1997

Y1 - 1997

N2 - Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle. For the serodiagnosis of this parasitic disease, two immunodominant clones from a bovine Neospora λgt11 library were identified, characterized, and expressed as recombinant proteins for the development of an enzyme-linked immunosorbent assay (ELISA). These two clones, designated N54 and N57, were 29 and 20 kDa, respectively, when expressed as histidine fusion proteins from the pRSET expression vector. Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecule; masses of 97, 87, 77, 67, and 64 kDa. Antibodies to recombinant protein N57 recognized four primary bands with molecular masses of 34, 31, 30, and 28 kDa. When a defined 'gold standard' panel of bovine Sera from confirmed Neospora-positive and Neospora-negative cattle were characterized by immunoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet. Binding to the N57 quadruplet was more variable. The same gold standard panel was used to evaluate and compare an N54-based ELISA, an N57-based ELISA, and a whole-tachyzoite lysate based ELISA. The sensitivities and specificities were 95 and 96% (N54 ELISA), 82 and 93% (N57 ELISA), and 74 and 93% (lysate ELISA). Thus, compared to the whole-tachyzoite lysate-based ELISA, both recombinant-protein-based ELISAs had higher sensitivities and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neosporosis.

AB - Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle. For the serodiagnosis of this parasitic disease, two immunodominant clones from a bovine Neospora λgt11 library were identified, characterized, and expressed as recombinant proteins for the development of an enzyme-linked immunosorbent assay (ELISA). These two clones, designated N54 and N57, were 29 and 20 kDa, respectively, when expressed as histidine fusion proteins from the pRSET expression vector. Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecule; masses of 97, 87, 77, 67, and 64 kDa. Antibodies to recombinant protein N57 recognized four primary bands with molecular masses of 34, 31, 30, and 28 kDa. When a defined 'gold standard' panel of bovine Sera from confirmed Neospora-positive and Neospora-negative cattle were characterized by immunoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet. Binding to the N57 quadruplet was more variable. The same gold standard panel was used to evaluate and compare an N54-based ELISA, an N57-based ELISA, and a whole-tachyzoite lysate based ELISA. The sensitivities and specificities were 95 and 96% (N54 ELISA), 82 and 93% (N57 ELISA), and 74 and 93% (lysate ELISA). Thus, compared to the whole-tachyzoite lysate-based ELISA, both recombinant-protein-based ELISAs had higher sensitivities and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neosporosis.

UR - http://www.scopus.com/inward/record.url?scp=0030781016&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030781016&partnerID=8YFLogxK

M3 - Article

VL - 4

SP - 692

EP - 699

JO - Clinical and Vaccine Immunology

JF - Clinical and Vaccine Immunology

SN - 1556-6811

IS - 6

ER -