Epitope mapping of anti‐troponin I monoclonal antibodies

Filatov, V.L.; Katrukha, Alexey G.; Bereznikova, A.; Esakova, Tatiana V.; Bulargina, T. V.; Kolosova, Olga V.; Северин, Е. С.; Gusev, Nikolai B.

doi:10.1080/15216549800203412

Cited by 24 publications

(44 citation statements)

References 6 publications

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“…The N-and C-terminal ends of human cTnI have been shown to be the strongest antigenic regions when mice were immunized with free cTnI (38 -40 ), whereas antibodies raised against the troponin complex often interact with the N-terminal region of cTnI (40 ). The human troponin autoantibodies, on the other hand, interfere with the binding of antibodies to the central region of cTnI (24 ).…”

Section: Discussionmentioning

confidence: 99%

Negative Interference in Cardiac Troponin I Immunoassays by Circulating Troponin Autoantibodies

et al. 2005

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Background: There are numerous potential sources of interference in immunoassays. Our aim was to identify the blood component that causes negative interference in cardiac troponin I (cTnI) immunoassays based on antibodies against the central part of cTnI. Methods: We isolated an interfering factor (IF) from a sample with low recovery of added cTnI, using several consecutive purification steps: caprylic acid precipitation, ammonium sulfate precipitation, and purification on Cibacron Blue gel and protein G columns. Purified IF was identified by gel electrophoresis and mass spectrometric analysis of protein bands. For the direct detection of human antibodies to cardiac troponin in serum samples, we developed immunoassays using three different anti-human immunoglobulin antibodies and measured troponin antibodies in samples with low and normal cTnI recovery. Results: Treatment with caprylic acid did not precipitate IF, but IF precipitated at 40% ammonium sulfate saturation. IF bound to a Cibacron Blue gel column, from which it was eluted with a linear salt gradient; it also bound to protein G. Gel electrophoresis of purified IF showed two major bands with molecular masses corresponding to the heavy (ϳ50 kDa) and light chains (ϳ25 kDa) of immunoglobulin, and their identities were confirmed by mass spectrometry. The presence of troponin-specific autoantibodies was confirmed in samples with low recoveries of cTnI by three different immunoassays. The median signals were significantly

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Section: Discussionmentioning

confidence: 99%

Negative Interference in Cardiac Troponin I Immunoassays by Circulating Troponin Autoantibodies

et al. 2005

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“…The entire N-domain of TnC subsequently undergoes a large tertiary conformational change, in which helices B and C move away as a unit from helices N, A, and D, exposing a buried hydrophobic pocket to the solvent (Ref. 5 and references within). The newly formed hydrophobic pocket is thought to allow the N-domain of TnC to interact with the C-terminal of TnI transferring the inhibitory domain of TnI away from actin (11).…”

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confidence: 99%

Mutations of Hydrophobic Residues in the N-terminal Domain of Troponin C Affect Calcium Binding and Exchange with the Troponin C-Troponin I96–148 Complex and Muscle Force Production

Davis

Rall

Alionte

et al. 2004

Journal of Biological Chemistry

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Interactions between troponin C and troponin I play a critical role in the regulation of skeletal muscle contraction and relaxation. We individually substituted 27 hydrophobic Phe, Ile, Leu, Val, and Met residues in the regulatory domain of the fluorescent troponin C F29W with polar Gln to examine the effects of these mutations on: (a) the calcium binding and dynamics of troponin C F29W complexed with the regulatory fragment of troponin I (troponin I 96 -148 ) and (b) the calcium sensitivity of force production. Troponin I 96 -148 was an accurate mimic of intact troponin I for measuring the calcium dynamics of the troponin C F29W -troponin I complexes. The calcium affinities of the troponin C F29W -troponin I 96 -148 complexes varied ϳ243-fold, whereas the calcium association and dissociation rates varied ϳ38-and ϳ33-fold, respectively. Interestingly, the effect of the mutations on the calcium sensitivity of force development could be better predicted from the calcium affinities of the troponin C F29W -troponin I 96 -148 complexes than from that of the isolated troponin C F29W mutants. Most of the mutations did not dramatically affect the affinity of calcium-saturated troponin C F29W for troponin I 96 -148 . However, the Phe 26 to Gln and Ile 62 to Gln mutations led to >10-fold lower affinity of calcium-saturated troponin C F29W for troponin I 96 -148 , causing a drastic reduction in force recovery, even though these troponin C F29W mutants still bound to the thin filaments. In conclusion, elucidating the determinants of calcium binding and exchange with troponin C in the presence of troponin I provides a deeper understanding of how troponin C controls signal transduction.Troponin C (TnC) 1 regulates striated muscle contraction and relaxation through the binding and release of Ca 2ϩ (for review see Refs. 1-3). Skeletal muscle TnC (ϳ18 kDa) consists of globular N-and C-terminal domains connected by a 31-residue ␣-helix (for review see Refs. 4 and 5). Both domains bind two Ca 2ϩ ions through a pair of EF hand Ca 2ϩ -binding motifs. Each pair of EF hands interacts with one another through a short antiparallel ␤-sheet connecting the two Ca 2ϩ -binding loops (Ref. 6 and references within). The EF hands are numbered I-IV, and the helices flanking the loops are designated A-H, with an additional N-terminal 14-residue ␣-helix (Fig. 1, N helix), which is absent in the closely related EF hand Ca 2ϩ -binding protein calmodulin.Much is known about the cation binding properties of TnC in solution. Each EF hand system binds Ca 2ϩ and Mg 2ϩ competitively, with the two C-terminal EF hands possessing higher Ca 2ϩ and Mg 2ϩ affinities (6 -8). In fact, the Ca 2ϩ -binding sites of the C-domain of TnC possess ϳ10-fold higher Ca 2ϩ affinity with a greater than 100-fold slower Ca 2ϩ dissociation rate compared with those in the N-domain (6, 9). In part because of its high Ca 2ϩ and Mg 2ϩ affinities and slow Ca 2ϩ exchange rates (as compared with the kinetics of muscle contraction and relaxation), the C-domain is thought to play a struc...

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“…TnT fulfills the role of anchoring the complex to tropomyosin and potentiating actomyosin ATPase activity (for reviews, see Refs. [1][2][3][4][5][6][7][8].…”

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confidence: 99%

Biological Function and Site II Ca2+-induced Opening of the Regulatory Domain of Skeletal Troponin C Are Impaired by Invariant Site I or II Glu Mutations

Pearlstone¹,

Chandra²,

Sorenson³

et al. 2000

Journal of Biological Chemistry

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Epitope mapping of anti‐troponin I monoclonal antibodies

Cited by 24 publications

References 6 publications

Negative Interference in Cardiac Troponin I Immunoassays by Circulating Troponin Autoantibodies

Negative Interference in Cardiac Troponin I Immunoassays by Circulating Troponin Autoantibodies

Mutations of Hydrophobic Residues in the N-terminal Domain of Troponin C Affect Calcium Binding and Exchange with the Troponin C-Troponin I96–148 Complex and Muscle Force Production

Biological Function and Site II Ca2+-induced Opening of the Regulatory Domain of Skeletal Troponin C Are Impaired by Invariant Site I or II Glu Mutations

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