16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals

S.K. Srivastava, V.J.B. Ruigrok, N.J. Thompson, A.K. Trilling, A.J.R. Heck, C.J.M. van Rijn, M.J. Beekwilder, M.A. Jongsma

Research output: Contribution to journalArticleAcademicpeer-review

5 Citations (Scopus)

Abstract

Background: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. Methodology/Principal Findings: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. Conclusions/Significance: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.
LanguageEnglish
Article numbere64040
JournalPLoS ONE
Volume8
Issue number5
DOIs
Publication statusPublished - 2013

Fingerprint

New World Camelids
llamas
Mycobacterium tuberculosis
Heat-Shock Proteins
heat shock proteins
Hot Temperature
epitopes
Epitopes
heat
antibodies
Antibodies
Sputum
sandwiches
Enzyme-Linked Immunosorbent Assay
enzyme-linked immunosorbent assay
antigens
Antigens
Electrospray ionization
Size exclusion chromatography
early diagnosis

Keywords

  • hiv-associated tuberculosis
  • immunological diagnosis
  • antibody fragments
  • mass-spectrometry
  • skin-test
  • antigen
  • complexes
  • responses
  • peptides
  • epitopes

Cite this

@article{e2271c2ff42c477c9b40d530e1d14bdc,
title = "16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals",
abstract = "Background: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. Methodology/Principal Findings: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. Conclusions/Significance: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.",
keywords = "hiv-associated tuberculosis, immunological diagnosis, antibody fragments, mass-spectrometry, skin-test, antigen, complexes, responses, peptides, epitopes",
author = "S.K. Srivastava and V.J.B. Ruigrok and N.J. Thompson and A.K. Trilling and A.J.R. Heck and {van Rijn}, C.J.M. and M.J. Beekwilder and M.A. Jongsma",
year = "2013",
doi = "10.1371/journal.pone.0064040",
language = "English",
volume = "8",
journal = "PLoS ONE",
issn = "1932-6203",
publisher = "Public Library of Science",
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}

16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals. / Srivastava, S.K.; Ruigrok, V.J.B.; Thompson, N.J.; Trilling, A.K.; Heck, A.J.R.; van Rijn, C.J.M.; Beekwilder, M.J.; Jongsma, M.A.

In: PLoS ONE, Vol. 8, No. 5, e64040, 2013.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - 16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals

AU - Srivastava, S.K.

AU - Ruigrok, V.J.B.

AU - Thompson, N.J.

AU - Trilling, A.K.

AU - Heck, A.J.R.

AU - van Rijn, C.J.M.

AU - Beekwilder, M.J.

AU - Jongsma, M.A.

PY - 2013

Y1 - 2013

N2 - Background: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. Methodology/Principal Findings: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. Conclusions/Significance: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.

AB - Background: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. Methodology/Principal Findings: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. Conclusions/Significance: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.

KW - hiv-associated tuberculosis

KW - immunological diagnosis

KW - antibody fragments

KW - mass-spectrometry

KW - skin-test

KW - antigen

KW - complexes

KW - responses

KW - peptides

KW - epitopes

U2 - 10.1371/journal.pone.0064040

DO - 10.1371/journal.pone.0064040

M3 - Article

VL - 8

JO - PLoS ONE

T2 - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 5

M1 - e64040

ER -