Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 3, pp. 349-363 © The Author(s) 2025. This article is an open access publication.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 3, pp. 386-402.

349

Development of Immunochemical Systems

for Detection of Human Skeletal TroponinI Isoforms

Agnessa P. Bogomolova

1,2,a

*, Ivan A. Katrukha

1,2,b

, Alexey M. Emelin

Artur I. Zabolotsky

, Anastasia V. Bereznikova

1,2

, Olga S. Lebedeva

Roman V. Deev

, and Alexey G. Katrukha

1,2

Lomonosov Moscow State University, Biological Faculty, 119234 Moscow, Russia

Hytest Ltd., 20520 Turku, Finland

Avtsyn Research Institute of Human Morphology, “Petrovsky National Research Centre of Surgery”,

117418 Moscow, Russia

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine

of Federal Medical-Biological Agency, 119435 Moscow, Russia

e-mail: bogomolova.agnessa@yandex.ru

e-mail: katrukhai@mail.ru

Received February 21, 2025

Revised March 7, 2025

Accepted March 11, 2025

Abstract— Troponin  I (TnI), together with troponin  T (TnT) and troponin  C (TnC), forms the troponin com-

plex, a thin filament protein of the striated muscle that plays a key role in regulation of muscle contraction.

In humans, TnI is represented by three isoforms: cardiac, which is synthesized only in myocardium, and

fast and slow skeletal, which are synthesized in fast- and slow-twitch muscle fibers, respectively. Skeletal TnI

isoforms could be used as biomarkers of skeletal muscle damage of various etiologies, including mechanical

trauma, myopathies, muscle atrophy (sarcopenia), and rhabdomyolysis. Unlike classical markers of muscle

damage, such as creatine kinase or myoglobin, which are also present in other tissues, skeletal TnIs are

specific for skeletal muscle. In this study, we developed a panel of monoclonal antibodies for immunochem-

ical detection of skeletal TnI isoforms using Western blotting (sensitivity: 0.01-1  ng per lane), immunohisto-

chemical assays, and fluorescence immunoassays. Some of the designed fluorescence immunoassays enable

quantification of fast skeletal (limit of detection [LOD]  =  0.07  ng/mL) and slow skeletal (LOD  =  0.1  ng/mL) TnI

isoforms or both isoforms (LOD  =  0.1  ng/ml). Others allow differential detection of binary (with TnC) or ternary

(with TnT and TnC) complexes, revealing composition of troponin forms in the human blood.

DOI: 10.1134/S0006297924601928

Keywords: troponin, fast skeletal troponinI, slow skeletal troponinI, biomarker, monoclonal antibodies, immu-

nochemical detection, immunoassays, skeletal muscle damage, myopathy, biomarker

* To whom correspondence should be addressed.

INTRODUCTION

Skeletal muscles accounts for up to 50% of the

total body weight in adults and are crucial for move-

ment, respiration, glucose and protein metabolism,

and thermogenesis. Therefore, skeletal muscle dis-

eases (including mechanical muscle damage, muscle

atrophy, and myopathies) could significantly affect

functioning of the entire body. Skeletal muscle damage

could be caused by trauma, surgery, intense physical

activity, or prolonged compression syndrome. Skeletal

muscle diseases (myopathies) are either inherited or

acquired. Inherited myopathies are mostly caused by

mutations in the genes of the contractile apparatus

and enzymes involved in carbohydrate and lipid me-

tabolism. The major reasons for acquired myopathies,

including inflammatory myopathies, are infections,

myotoxic drugs, and various diseases. Muscle atrophy

is often observed in the patients immobilized due to

prolonged hospitalization and those with limb dener-

vation, cancer, chronic heart failure, or sarcopenia

[1, 2]. Specific and highly sensitive tools for detecting

BOGOMOLOVA et al.350

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

muscle fiber damage are required to successfully treat

skeletal muscle diseases and/or prevent skeletal mus-

cle degeneration [3, 4]. A common method for diag-

nosing skeletal muscle damage is immunochemical

detection of cytoplasmic proteins released into the

bloodstream during muscle fiber necrosis. Creatine

kinase and myoglobin are typically used as such bio-

markers [5-7]. However, apart from the skeletal mus-

cle, these proteins are synthesized in other tissues,

which reduces specificity of the diagnosis. Creatine

kinase is represented by three isoenzymes – MM,

MB, and BB. Their ratio is 98% MM and 2% MB in

the skeletal muscles and 70-80% MM and 20-30% MB

in the heart (BB is present mainly in the brain) [6].

Myoglobin is synthesized in both skeletal muscles and

heart [7]. Isoforms of skeletal troponin  I (TnI), which

are detected only in the skeletal muscles, could serve

as alternative and more specific biomarkers.

TnI is a contractile apparatus protein in skele-

tal and cardiac muscles. Together with troponin  T

(TnT) and troponin  C (TnC), it forms a noncovalent

complex with molar ratio 1  :  1  :  1 and participates in

the Ca

-dependent regulation of muscle contraction.

In humans, TnI is represented by three tissue-specif-

ic isoforms: cardiac (cTnI), which is synthesized only

in myocardium; fast skeletal (fsTnI) and slow skeletal

(ssTnI) isoforms, which are specific to fast-twitch and

slow-twitch muscle fibers, respectively. The degree of

similarity of skeletal TnI isoforms is ~60%, with the

C-terminal parts being the most conserved parts of

the molecules [8].

In the series of studies on drug myotoxicity con-

ducted in rats to determine sensitivity and specificity

of skeletal TnI isoforms, this marker outperformed cre-

atine kinase not only in specificity, but also in sensitiv-

ity and diagnostic accuracy [9]. In humans, concentra-

tion of the skeletal TnI isoforms in the blood increases

after intense physical exercise, rhabdomyolysis, vari-

ous traumas, muscular dystrophies, and inflammatory

myopathies, suggesting that these markers could be

used to diagnose these conditions [9-16]. The skeletal

TnI isoforms could also be used to assess severity of

the course of muscular dystrophies and to monitor re-

sponse to the corticosteroid supportive therapy, along

with other developing treatment methods [17].

Some conditions are characterized by selective

damage to certain types of muscle fibers. For instance,

administration of statins predominantly induces inju-

ry to the fast-twitch fibers, whereas administration of

fibrates mostly affects the slow-twitch fibers [3,  18].

Eccentric muscle contractions primarily lead to the

fast-twitch fiber deterioration, which is accompanied

by increase in the fsTnI concentration in blood [15,

19]. Denervation or limb immobility, spinal cord inju-

ry, prolonged bed rest, and exposure to microgravity

usually induce the slow-to-fast fiber type shift, where-

as sarcopenia, cachexia, starvation, and glucocorticoid

administration result in the fast-to-slow fiber shift [20].

Therefore, methods that can specifically detect injury

in the particular type of fibers could provide addi-

tional possibilities for differential diagnosis of some

diseases. Thus, it is important to develop methods that

simultaneously detect both skeletal TnI isoforms and

can distinguish between fsTnI and ssTnI.

Immunochemical methods provide a convenient

approach for detecting skeletal TnIs. Among these,

Western blotting (WB) is suitable for qualitative and

quantitative detection of skeletal TnI isoforms in cell

and tissue lysates. Immunocytochemical or immuno-

histochemical (IHC) staining is performed to visual-

ize proteins in the samples of fixed cells and tissues.

Enzyme-linked immunosorbent assay (ELISA) and

fluorescence immunoassay (FIA) are commonly used

for quantitative measurement of antigens in blood

to diagnose skeletal muscle injury. cTnI, which has

been thoroughly examined using immunochemical

methods, is released into the bloodstream not in a

free form but as a complex with TnT and TnC, and is

present in blood as part of the ternary or binary (with

TnC) complexes [21-23]. To the best of our knowl-

edge, there are no experimental data on the release

of skeletal TnI forms. However, because of homology

between the cardiac and skeletal troponins, it could be

assumed that the latter are also present in the blood

as complexes. Therefore, some epitopes of the skel-

etal TnI could be screened by TnT and TnC, which

should be considered when developing diagnostic sys-

tems. Future research should focus on the assays that

recognize total skeletal TnI, as well as methods that

can differentiate between the various forms of fsTnI

and ssTnI in blood.

In this study, we describe both approaches: as-

says that can detect fsTnI and ssTnI both separately

and together in free form or in complex (binary or

ternary), and methods that can differentially identi-

fy these complexes. Aim of this study was to obtain

and characterize monoclonal antibodies (mAbs) and

develop different immunochemical detection systems

based on WB, IHC, and FIA for determination of the

skeletal TnI isoforms.

MATERIALS AND METHODS

All reagents were purchased from Sigma-Aldrich

(USA), unless stated otherwise. The following recom-

binant proteins (expessed in Escherichia coli) were

used: fsTnI, fast skeletal TnT (fsTnT), fast skeletal

TnC (fsTnC), ssTnI, slow skeletal TnT (ssTnT), slow

skeletal/cardiac TnC (ss/cTnC), and cTnI. The follow-

ing complexes were used: fast skeletal TnI-TnC com-

plex (fsIC), fast skeletal TnI–TnT–TnC complex (fsITC),

IMMUNOCHEMICAL DETECTION OF HUMAN SKELETAL TnI 351

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

slow skeletal TnI–TnC complex (ssIC), cardiac TnI–TnC

complex (cIC), and cardiac TnI–TnT–TnC complex

(cITC). Monoclonal antibodies (mAbs) specific toward

cTnI (560, 4C2), TnT (TnT111), and fsTnC as part of

the fsITC (TnC99A5) were from Hytest (Finland). Anti-

MyHC1 mAb (antibodies specific to MHC) were pur-

chased from Abcam (UK).

Generation of mAbs. Female BALB/c mice

(“Nursery for laboratory animals”, IBCh RAS) weighing

20g 6-8 weeks old at the start of the experiment were

used to obtain mAbs. The mice were housed in open-

type conventional cages with free access to food and

water. Isoflurane inhalation was used to euthanize

mice. All procedures were conducted in compliance

with the European Community Directive 2010/63/EU.

Mice were immunized with recombinant fsTnI, fsITC,

or ssTnI. Spleen cells from the immunized mice were

hybridized with sp 2/0 myeloma cells using a stan-

dard procedure [24]. Indirect ELISA was used to select

clones that produced antibodies specific to fsTnI or

ssTnI.

Indirect ELISA. For this assay, solutions of 25-

100  ng of antigen in a phosphate-buffered saline (PBS;

10  mM  KH

, 150  mM  NaCl, pH  7.4) were placed

into 96-well polystyrene plates (Greiner, Germany) for

sorption, and incubated for 40  min at 25°C with con-

stant stirring. The wells were next washed to remove

nonspecifically adsorbed components using a PBS

containing 0.1%  Tween  20 (PBST). Next, 0.5-1  μg/mL

of antibodies in PBST were added. Serial dilutions

were performed if necessary. The plates were further

incubated for another 40  min at 25°C with constant

stirring. Following incubation, they were washed four

times and 100  μL of a solution of detection goat poly-

clonal antibodies targeting mouse IgG conjugated with

horseradish peroxidase (Beckman Dickinson, USA)

were added. After another round of incubation for

20  min at 25°C with constant stirring, the plates were

washed with PBST six times. This was followed by ad-

dition of 100  μL of a substrate (3,3′,5,5′-tetramethyl-

benzidine in 0.1  M  Na-acetic buffer, pH  4.5, containing

0.01%  H

). After color developed (within 10  min or

less), the reaction was stopped by adding 25  μL of 2  M

orthophosphoric acid per well, and absorbance in the

wells was measured at 450 nm using a Multiscan EX

plate reader (Labsystems).

Epitope mapping of mAbs. Specificity of the

mAbs toward epitopes was determined using an in-

direct ELISA with peptides conjugated with a car-

rier protein. A set of 20 amino acid peptides that

comprised the entire fsTnI or ssTnI sequence, with

an overlap of six amino acids, was synthesized by

GenScript (USA). An additional cysteine residue was

added to the C-terminus of each peptide (except for

the last peptide, where the Cys residue was added to

the N-terminus) for conjugation with a carrier protein

(ovalbumin or bovine serum albumin). If the antibody

recognized one peptide, the epitope of the antibody

was considered to be this peptide. If the antibody

recognized two neighboring peptides, the overlapping

site was considered the epitope.

Sandwich FIA with europium chelate. FIA was

performed according to the standard procedures [25,

26]. Solutions of mAbs in PBS were adsorbed onto

96-well polystyrene plates (Greiner, Germany) at con-

centration 2  μg/mL (50  μL per well) and incubated for

40  min at 25°C with constant stirring. The wells were

then washed with a washing buffer (10  mM Tris-HCl,

pH  7.8, 0.9%  NaCl, 0.025%  Tween  40, 0.05%  NaN

) on a

PlateWasher (Perkin Elmer, USA) to remove non-specif-

ically adsorbed components. After washing, 25  μL of a

solution of detection mAbs conjugated with stable eu-

ropium chelate in 50  mM  Tris-HCl pH  7.8, 0.9%  NaCl,

0.5%  bovine serum albumin (BSA), 0.01%  Tween  40,

0.5%  NaN

and, optionally, 20  mM  EDTA, along with

25  μL of antigen diluted in 150  mM  KCl, 20  mM  Tris-

HCl, 7.5%  BSA, and 0.15%  NaN

were added into each

well. The resulting mixture was incubated for 40  min

at 25°C with stirring, and the wells were washed

six times with a washing buffer. After that, 100  μL

of enhancement solution (0.1  M  CH

COOH, pH  3.2,

50  μM trioctylphosphine oxide, 50  μM 4,4,4-trifluoro-

1-(2-naphthyl)-1,3-butanedione, 0.1%  Triton  X-100) was

added into each well incubated for 10  min under

constant stirring. Phosphorescence intensity was next

measured at an excitation wavelength of 340  nm and

emission wavelength of 615  nm using a Victor 1420

Multilabel Counter (Perkin Elmer).

Sensitivity parameters of FIAs. Limit of detec-

tion (LOD) was calculated as:

LOD  =  mean

blank

  + 1.645×SD

blank

  + 1.645×SD

low  concentration

(where mean

blank

is mean background signal, SD

blank

is standard deviation of the background signal, and

low  concentration

is standard deviation of the sample

with low concentration of the analyte). Samples of

fsIC and ssIC were used for calibration. To deter-

mine LOD, background signal was measured in 36

repetitions, while samples with low analyte concen-

trations (selected from the [mean

blank

  +  1.645×SD

blank

]

to 4×[mean

blank

  +  1.645×SD

blank

]) were measured in

60 repetitions. This experiment was repeated three

times and mean value was calculated. Outliers were

identified using the formula: mean  ±  2×SD. Data are

presented as mean ± SD.

Linearity range of FIAs. To construct linear

approximations, a series of dilutions of the target

antigen were made in the concentration range 0.04-

1280  ng/mL. Linear range was determined accord-

ing to the CLSI EP6-A procedure (Evaluation of the

Linearity of Quantitative Measurement Procedures:

BOGOMOLOVA et al.352

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

A statistical Approach; Approved Guideline) [27] using

Microsoft Office Excel 2007. Linear approximations

were calculated using the “Data Analysis” add-in and

the “Regression” function, which was employed to

perform extrapolation using third-order, second-order,

and first-order polynomials. Linear range was defined

as the region where difference between the values

predicted by the polynomial and those predicted by

the linear model were less than 5% for all data points.

Cross reactivity of FIAs with other TnI iso-

forms. Cross-reactivity with other TnI isoforms was

determined using sandwich FIA. Calibration curve was

constructed using a series of antigen dilutions at con-

centrations ranging from 0.3 to 80  ng/mL. Additional-

ly, dilutions of cross-reacting antigens were prepared

at concentrations of 2000  ng/mL and 1000  ng/mL.

Cross-reactivity (%) was calculated using the formula

Ag  real

  ×  100, where C

is antigen concentration

calculated from the calibration curve, and C

Ag  real

the real antigen concentration.

Western blotting. For immunochemical staining

in WB, proteins were separated via sodium dodecyl

sulfate-polyacrylamide gel electrophoresis under re-

ducing conditions. Next, the proteins were blotted

onto a 0.45  μm nitrocellulose membrane (Bio-Rad,

USA) at a constant voltage of 100  V for 40 min. The

membrane was blocked with PBST containing 5%

skim milk and incubated overnight at 4°C in 10  mL

of PBST containing 5%  skim milk and biotin isothiocy-

anate-conjugated mAbs (1  μg/mL). After washing with

PBST, the membrane was incubated with streptavi-

din-polyperoxidase (Thermo Fisher Scientific, USA)

in PBST for 40  min at room temperature and washed

again with PBST. Immune complexes were detected

using a SuperSignal West Femto Maximum Sensitivity

Substrate (Thermo Fisher Scientific) with a ChemiDoc

MP Imaging System (Bio-Rad). The bands were ana-

lyzed using ImageLab  6.1 software (Bio-Rad).

Gel filtration. Proteins were separated using an

AKTA Pure chromatography system (GE HealthCare,

USA) equipped with a Hiload Superdex 200 16/600  pg

column (Cytiva, USA). Gel filtration buffer consisted of

50  mM  Tris-HCl, 150  mM  NaCl, 5  mM  CaCl

, 0.1%  BSA,

and 0.1%  NaN

. The column was loaded with a

100-ng/mL recombinant antigen solution (1  mL) or

2.5  mg skeletal muscle tissue extract diluted in a gel

filtration buffer (1  mL). Flow rate was set at 1  mL/min.

The resulting fractions were analyzed using sandwich

FIA with different mAb pairs.

Preparation of tissue sections and immunohis-

tochemistry. To prepare tissue sections, tissue blocks

from the archive of the Department of Pathological

Anatomy of the I.  I.  Mechnikov Northwestern State

Medical University of Russia were used. Heart blocks

of human fetuses that died from causes unrelated to

cardiac pathology at 29 and 41-42 weeks of intrauter-

ine development, adult myocardium samples of the

anterior wall of the left ventricle, and adult m. vastus

lateralis were included. All tissue samples were collect-

ed according to the protocol approved by the local Eth-

ical Committee of the Lopukhin Federal Research and

Clinical Center of Physical-Chemical Medicine at the

Federal Medical Biological Agency of Russia on 06Feb-

ruary 2024 (meeting protocol number, 2024/02/06).

Before archiving, the samples were subjected to a

standard histological procedure: fixation in a neutral

10%  formalin solution, dehydration in isopropanol,

embedding in paraffin, and subsequent block prepa-

ration [28]. Histologic sections of 3-μm thickness were

prepared using a Thermo Fisher HM 325 rotary mi-

crotome.

Tissue sections were de-paraffinized and rehy-

drated, then subjected to heat-induced antigen retriev-

al in a sealed container using a Trilogy® solution (Cell

Marque, USA). The process was carried out in a dry

heat cabinet for 12  h at 60°C. After washing with dis-

tilled water, a 3% solution of hydrogen peroxide was

added to inhibit activity of endogenous peroxidases.

The tissue sections were next washed with distilled

water and PBS, treated with donkey serum solution

(up to 5% v/v; Jackson ImmunoResearch, USA) in PBS,

and incubated for 30  min. In the next step, the serum

solution was removed from the tissue sections, prima-

ry mAbs were added, and the sections were placed

in a humid chamber in a dry heat cabinet at 37°C

for 2 h. Primary mAbs specific to troponins were di-

luted in an Antibody Diluent (Abcam) at a ratio of

1  :  100, anti-MyHC antibodies at 1  :  4000, and anti-

MyHC1 antibodies at 1  :  500. After 2  h, the tissue slices

were washed three times in PBS and treated with a

N-Histofine® Simple Stain™ Max PO (Nichirei Biosci-

ences, Japan) for 30  min at 25°C. The tissue sections

were washed again in PBS and a diaminobenzidine

(DAB) substrate kit (Abcam) was applied. The slices

were stained with a Mayer’s haematoxylin solution,

dehydrated, clarified in xylene, and placed under a

coverslip.

In a negative control incubation with primary

mAbs specific to the target proteins was omitted from

the IHC protocol to evaluate nonspecific binding. For

all antibodies, two independent staining were per-

formed for each sample.

Human skeletal muscle extract preparation. To

50-65  mg of a skeletal muscle tissue sample, 1  mL of

an extraction buffer (50  mM  Tris-HCl, pH  7.5, 0.4  M

LiCl, 5  mM  CaCl

, phenylmethylsulphonyl fluoride,

pepstatin  A, and leupeptin) was added. The tissue was

homogenized and proteins were extracted for 30  min

on ice. The mixture was next centrifuged for 10  min

at 4°C and 20,000g. The resulting supernatant, the

skeletal muscle tissue extract, was used for further

analysis.

IMMUNOCHEMICAL DETECTION OF HUMAN SKELETAL TnI 353

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

Statistics. Statistical analysis and construction

of graphs were carried out using Microsoft Office

Excel 2007.

RESULTS

Development and characterization of mAbs.

A panel of 97 mAbs that recognize fsTnI, ssTnI, or

both isoforms simultaneously was obtained using the

hybridoma technique. Indirect ELISA was utilized for

primary antibody selection, and mAbs that interact-

ed with only one TnI isoform (without cross-reactivi-

ty with other isoforms) or both skeletal TnI isoforms

were identified. According to the epitope mapping

results, mAbs recognizing regions 30-105  aa and 156-

175  aa (specific to fsTnI), 2-119  aa and 142-187  aa (spe-

cific to ssTnI), and 30-49  aa and 156-182  aa (specific

to both skeletal TnI isoforms) were obtained. These

mAbs were tested in pairs using FIA, and combina-

tions capable of detecting the target antigen with the

highest sensitivity were selected. To select mAbs that

were suitable for WB, we used the following criteria:

specificity to the exact TnI isoform in WB (i.e., no

cross-reactivity with other TnI isoforms), no interac-

tion with non-target proteins in the skeletal muscle

extract or serum, and sensitivity in WB. The mAb

epitopes selected for further studies are shown

in Fig.  1.

Specificity of mAbs in WB. The mAbs with the

highest sensitivity and specificity for the target pro-

tein in WB were selected (Fig.  2). Sensitivity of the

Fig.  1. TnI isoforms. Alignment of the three TnI isoforms – fsTnI (Expasy identificator P48788), ssTnI (P19237), cTnI

(P19429)– was performed using Clustal Omega. Identical amino acids residues are marked in dark grey; residues identical

in two isoforms are marked in light grey. Ovals indicate TnT- and TnC-binding sites (approximate numbering of residues

is for the fsTnI sequence). Rectangles indicate the antibody epitopes. Footnotes indicate the name of the mAb, approximate

boundaries of its epitope, and its specificity. If the obtained antibody is specific to both skeletal TnI isoforms, the epitope

is indicated for the molecule that was used as an immunogen when obtaining this mAb [29-38].

Fig.  2. Recognition of different TnI isoforms by mAbs. a) skTnI89 (fsTnI), b) skTnI38 (ssTnI), c) skTnI50 (fsTnI and ssTnI),

d) 560 (cTnI). Lanes: 1) cTnI, cTnT, ss/cTnC in 1  :  1  :  1 molar ratio, cTnI is 67 ng/lane; 2) ssTnI, ssTnT, ss/cTnC in 1  :  1  :  1

molar ratio, ssTnI is 67ng/lane; 3) fsTnI, fsTnT, fsTnC in 1  :  1  :  1 molar ratio, fsTnI is 67 ng/lane; 4) human skeletal muscle

tissue extract (m. vastus lateralis), 42 µg of tissue/lane.

BOGOMOLOVA et al.354

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

Fig.  3. Immunochemical detection of skeletal TnI isoforms in human striated muscle tissues. mAbs used for IHC staining:

a)skTnI15 (fsTnI), b)skTnI30 (ssTnI), c)skTnI50 (fsTnI  +  ssTnI), d)4C2 (cTnI). 1)Adult skeletal muscle (m.vastus lateralis);

2) 29-week fetal heart; 3) 41-42-weeks fetal heat; 4) adult heart.

skTnI89 mAb in WB was 0.01  ng fsTnI/lane and

skTnI38 was 0.11  ng ssTnI/lane (Fig.  S1 in the Online

Resource

  1). These mAbs did not interact with cardiac

or other skeletal isoforms (Fig.

  2, a andb). Sensitivity

of the skTnI50 mAb, which is specific to fsTnI and

ssTnI, was 1  ng fsTnI/ssTnI per lane (Fig.  S1 in the On-

line Resource 1). This mAb did not interact with cTnI.

Methods to detect skeletal TnI isoforms in

tissues. Skeletal muscle tissue sections were subject-

ed to IHC staining with mAbs specific toward fsTnI

(skTnI15), ssTnI (skTnI30), and both skeletal TnI iso-

forms (skTnI50). Accumulation of DAB (intensity of

staining) was observed in the skeletal muscle fibers

(sarcoplasmic staining pattern) but not in the adult

human cardiomyocytes (Fig. 3). Utilization of skTnI15

and skTnI30 resulted in accumulation of DAB only in

some of the fibers, whereas using skTnI50 resulted

in positive staining reactions in all fibers. To identify

IMMUNOCHEMICAL DETECTION OF HUMAN SKELETAL TnI 355

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

Fig.  4. Immunochemical detection of skeletal TnI and MHC isoforms in human skeletal muscle. mAbs used for IHC staining:

a) skTnI15 (fsTnI), b) skTnI30 (ssTnI), c) anti-MyHC1, d) anti-MyHC.

the fast- and slow-twitch muscle fibers, we performed

tissue staining with the anti-MyHC antibodies specif-

ic to the myosin heavy chain (MHC) isoform, which

is present only in the slow-type muscle fibers, and

with the anti-MyHC1 antibodies specific to the MHC

isoform, which is synthesized in the fast-type muscle

fibers. skTnI30 stained the same fibers as anti-MyHC,

while skTnI15 stained the same fibers as anti- MyHC1,

indicating specificity of skTnI30 and skTnI15 towards

slow- and fast-twitch muscle fibers, respectively

(Fig. 4).

ssTnI is expressed in heart during the prenatal

period and switches to cTnI during the postnatal pe-

riod [39]. IHC staining with skTnI30 (specific to ssT-

nI) led to DAB accumulation in the fetal hearts at 29

and 41-42 weeks of prenatal development, but not in

the adult hearts (Fig.  3b). skTnI15 (fsTnI) was neither

detected in cardiomyocytes of mAb fetal hearts nor

in the adult myocardium (Fig.  3a). skTnI50 (specific

to both fsTnI and ssTnI) did not interact with either

the fetal or adult myocardium (Fig.  3c). We presume

that absence of the staining of the fetal myocardi-

um by mAb skTnI50 could be due to insufficient

sensitivity of this antibody (Fig.  S1 in the Online

Resource 1).

Development of sandwich FIAs to detect skel-

etal TnI isoforms in human blood. Three immu-

nochemical assays were developed based on the ob-

tained mAbs for the detection of fsTnI, ssTnI, or both

skeletal TnI isoforms. Table  1 lists analytical param-

eters of the FIAs (see also Fig.  S2 in the Online Re-

source 1).

The developed assays showed reduced recogni-

tion of IC and ITC compared to their recognition of

free TnI. Therefore, to restore intensity of the immu-

nochemical signal, EDTA was added to the solution.

Table 1. Sandwich FIAs to detect skeletal TnI isoforms

Skeletal TnI isoforms LOD, ng/mL (mean ± SD) Linear range, ng/mL Level of cross reactivity, %

skTnI89-skTnI91 (fsTnI) 0.07 ± 0.02 0.08-80 <0.004% for ssTnI and cTnI

skTnI27-skTnI58 (ssTnI) 0.10 ± 0.02 0.31-640 <0.005% for fsTnI, 0.026% for cTnI

skTnI25-skTnI50

(fsTnI and ssTnI)

0.10 ± 0.02 for fsTnI

0.11 ± 0.03 for ssTnI

0.08-320 (fsTnI)

0.31-320 (ssTnI)

<0.005% for cTnI

BOGOMOLOVA et al.356

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

Fig.  5. Detection of free TnI, IC, and ITC with sandwich FIAs in the absence and presence of EDTA. a) skTnI89-skTnI91;

b)skTnI58-skTnI27; c,d) skTnI25-skTnI50. Recombinant ssITC was not used due to its instability (dissociation into ssIC and

free TnT). a, c) Curves: 1) fsTnI in the absence of EDTA, 2) fsTnI in the presence of EDTA, 3) fsIC in the absence of EDTA,

4) fsIC in the presence of EDTA, 5) fsITC in the absence of EDTA, 6) fsITC in the presence of EDTA. b, d) Curves: 1) ssTnI

in the absence of EDTA, 2)ssTnI in the presence of EDTA, 3)ssIC in the absence of EDTA, 4) ssIC in the presence of EDTA.

Fig.  6. Detection of troponins in human skeletal muscle ex-

tract via sandwich FIA in the absence and presence of EDTA.

Fractions obtained from skeletal muscle extract after gel fil-

tration on the Superdex 200 16/600 column were analyzed

using the skTnI58-skTnI27 assay via sandwich FIA in the

absence (1) and presence (2) of EDTA. Elution volumes of

ssIC and ssITC standards are indicated by arrows.

Binding of the proteins within the troponin complex

is Ca

-dependent, and previous studies on cardiac

isoforms have shown that addition of EDTA leads to

dissociation of the ternary cITC and binary cIC; this

results in restoration of immunochemical signal in the

FIAs based on antibodies specific to free TnI [14, 21,

40]. Additionally, according to our results, incubation

of fsITC and fsIC with EDTA results in the complete loss

of immunochemical activity in the skTnI14-TnC99A5

assay (see next paragraph), indicating dissociation of

fsITC and fsIC (Fig.  S3 in the Online Resource  1). More-

over, when fsITC was incubated in the buffer solution

containing different concentrations of Ca

and EDTA,

and samples were analyzed at different time intervals

in the skTnI89-skTnI91 assay (Fig.S3 in the Online Re-

source 1), we observed minimal increase in the signal

in the presence of Ca

, and maximal increase in the

presence of EDTA. This could indicate both conforma-

tional changes and dissociative processes that lead to

epitope unveiling. Therefore, we hypothesized that ad-

dition of EDTA to the developed FIAs led to a confor-

mational change or dissociation of the complex and,

consequently, to recovery of immunochemical activity

in detection of the skeletal TnI isoforms (Figs.  5 and  6).

After addition of EDTA, immunochemical signals

of the skTnI89-skTnI91 (Fig.  5a, curves  5  and  6) and

skTnI25-skTnI50 (Fig.  5c, curves 3-6; Fig.  5d, curves

3 and 4) assays were recovered in the samples con-

taining binary or ternary complexes. The recombi-

nant ssITC (slow skeletal ITC) obtained in vitro was

unstable and dissociated into ssIC and free TnT;

hence, the recombinant protein cannot be analyzed

with the aforementioned method. Therefore, we stud-

ied activity of the skTnI58-skTnI27 assay using hu-

man skeletal muscle extracts after separation by gel

filtration. When analyzing chromatography profile

using this assay, we observed increase in the signal

of fractions contain ing endogenous ssITC after EDTA

addition (Fig. 6).

Sandwich FIAs to detect different forms of TnI.

An assay capable of recognition of only fsTnI and fsIC,

skTnI89-skTnI1, was selected to study the fsTnI forms.

IMMUNOCHEMICAL DETECTION OF HUMAN SKELETAL TnI 357

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

Fig.  7. Specificity of sandwich FIAs for fsTnI (a, b) and ssTnI (c, d) form analysis. a) skTnI89-skTnI1; b) skTnI14-TnC99A5;

c)skTnI58-skTnI27; d)TnT111-skTnI38. a, b)Curves: 1)fsTnI, 2) fsIC, 3)fsITC; c)curves: 1)ssTnI, 2)ssIC; d)curves: 1)skel-

etal muscle extract (the source of ssITC), 2) ssIC, 3) ssTnT, 4) fsITC, 5) fsTnT.

Fig.  8. Forms of skeletal TnI isoforms in human skeletal muscle extract. Immunoreactivity profile of the fractions obtained

with gel filtration (using Superdex200 16/600 column), analyzed via sandwich FIAs using different antibody pairs. a)Sand-

wich FIAs for detecting fsTnI forms; curves: 1) skTnI89-skTnI1, 2) skTnI14-TnC99A5; b) Sandwich FIAs for detecting ssTnI

forms; curves: 1) skTnI58-skTnI27, 2) TnT111-skTnI38.

Cross-reactivity with fsITC was ~4% (Fig.  7a). When

analyzing the fractions obtained from gel filtration

of the skeletal muscle extract, only fsIC was detect-

ed, as there was no free fsTnI in the extract (Fig.  8a).

The second antibody pair, skTnI14-TnC99A5, predomi-

nantly interacted with fsITC (Fig.  7b). Cross-reactivity

of this assay with free fsTnI was ~4%, and that with

fsIC was ~37%. When analyzing fractions obtained

after gel filtration of the skeletal muscle extract, the

skTnI14-TnC99A5 assay mediated detection of fsITC

(Fig.  8a, curve  2) with an elution volume of ~62  mL.

In the process, cross-reactivity with fsIC did not inter-

fere with identification of fsITC because these com-

plexes had different elution volumes (Fig. 8a).

For the ssTnI forms, two assays were selected:

the TnT111-skTnI38 assay, which recognizes only ssITC

and the skTnI58-skTnI27 assay, which recognizes all

forms of the slow skeletal isoform – ssTnI, ssIC, and

ssITC (Fig.  7, c, d). Upon analysis of the fractions of

skeletal muscle extract produced with gel filtration,

these pairs enabled differential detection of ssIC and

ssITC (Fig. 8b).

DISCUSSION

Immunochemical detection of skeletal TnI iso-

forms is of great importance as they are skeletal

BOGOMOLOVA et al.358

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

muscle- specific proteins and are biomarkers of skel-

etal muscle pathologies. In the present study, we ob-

tained a panel of mAbs that specifically recognized

the skeletal TnI isoforms, fsTnI, ssTnI, or both. We

selected the mAbs with the highest sensitivity and

specificity that were suitable for detection of the iso-

forms across various applications (see Table S1 in the

Online Resource 1).

Troponins are known to undergo post-translation-

al modifications such as phosphorylation. In particu-

lar, the fsTnI isolated from rabbit skeletal muscle is

in a partially phosphorylated form [41-43]. However,

based on the analysis of the existing data, we could

speculate that the proportion of phosphorylated forms

of skeletal TnI in the blood is negligible [44-46], and,

therefore, would not significantly affect skeletal tro-

ponins detection by antibodies [8]. Thus, recombinant

fsTnI and ssTnI expressed in E.coli without additional

modifications were selected as suitable immunogens

for mAb production.

The mAbs that specifically bind to skeletal TnI

isoforms in WB were selected. Sensitivity of the devel-

oped methods allows reliable detection of the target

proteins in tissue samples. Specifically, sensitivity of

detection with skTnI89 (fsTnI) is 0.01ng/lane, skTnI38

(ssTnI) is 0.11  ng/lane, and skTnI50 (fsTnI and ssTnI)

is 1  ng/lane, while the TnI content in tissue was esti-

mated to be 20  mg per 100  g of tissue (data were ob-

tained for cardiac TnI isoform) [47]. Thus, sensitivity

of these mAbs in WB is sufficient to detect skeletal TnI

isoforms in approximately 0.05-5  μg of muscle tissue.

The mAb specific to fsTnI (skTnI89) recognizes cen-

tral region of the molecule, which allows detection of

not only the full-length protein but also its proteolytic

fragments. The mAbs skTnI58 (specific to ssTnI) and

skTnI50 (recognizes both skeletal TnI isoforms), which

are specific to the C-terminal regions of the molecules,

allow detection of the full-length proteins and skeletal

TnI isoforms that are proteolytically cleaved at their

N-terminal regions.

The developed mAbs could be used for IHC de-

tection of the skeletal troponins; they effectively rec-

ognize skeletal muscle fibers but do not stain adult

cardiomyocytes. Specificity of the skTnI15 and skTnI30

toward fast- and slow-twitch fibers, respectively, was

confirmed by typing skeletal muscle sections with

mAbs that interact with different isoforms of MHC.

This is a common method to distinguish muscle fiber

types [48]. Thus, our mAbs could be used as an alter-

native method for muscle-fiber typing. During prena-

tal development, ssTnI is predominantly expressed in

the heart and is completely replaced by cTnI during

the postnatal period [39]. Our data are consistent with

this finding: the mAb skTnI30 recognized ssTnI in the

tissue sections of human fetal hearts (29-week- and

41-42-week-old) but did not stain adult cardiomyo-

cytes. Thus, it could also be possible to use this mAb

for specific detection of ssTnI in the non-terminally

differentiated human cardiomyocytes to determine

the extent of cardiac tissue maturation. To date, var-

ious approaches for heart transplantation have been

developed, including generation of cardiac tissue from

the donor cells using induced pluripotent stem cell

(iPSC) technology. However, reaching maturity is a

challenge for these cardiomyocytes. Many researchers

have recognized the need for a differential marker

and considered the ratio of cTnI and ssTnI as a poten-

tial candidate [49-51]. Currently, WB is used to assess

the degree of maturity, but only cTnI has been detect-

ed in the cells and tissues. However, the ssTnI-specific

mAbs could be combined with the cTnI-specific mAbs

to determine the cTnI : ssTnI ratio in non-terminally

differentiated human cardiac muscle tissue and the

degree of maturity.

One of the most common diagnostic methods is

measurement of biomarkers in biological fluids, includ-

ing blood. Basal concentration of the skeletal TnI iso-

forms can reach the values of the tenth of nanograms

to nanograms per mL [11, 13, 14], and their increased

levels in blood indicate skeletal muscle fiber damage.

cTnI is released into the bloodstream and is present in

the blood not in its free form but as part of various com-

plexes with cTnT and ss/cTnC: ternary complexes with

cTnT and ss/cTnC and binary complexes with ss/cTnC.

The ratio of these forms changes over time [21, 52-54].

Therefore, detection of cTnI in all its possible forms

(complexes) is considered to be the most reliable ap-

proach. To the best of our knowledge, there are no

experimental data describing the forms in which the

skeletal TnIs are released into the human bloodstream

or how their composition and ratio change over time.

In the present study, we developed immunoassays that

differentially interact with the free skeletal troponins

and troponins within complexes. For fsTnI, we de-

signed an assay that could detect free fsTnI and fsIC

but not fsITC (skTnI89-skTnI1), along with the anti-

body pair that interacted predominantly with fsITC

(skTnI14-TnC99A5) but not with the binary complex

or free fsTnI. For ssTnI, the assay that interacts with all

forms of the protein was developed (skTnI58-skTnI27),

along with the antibody pair that only detects ssITC

(TnT111-skTnI38). Combination of these FIAs with the

preliminary separation by gel filtration allows differ-

ential detection of various forms of skeletal TnIs and

could be further used to analyze troponin composi-

tion in the blood samples. It would be interesting to

compare the data on the content of skeletal troponin

complexes in blood with the extensively researched

dynamics of cTnI. Moreover, the ratio of skeletal

TnI isoforms could vary in different skeletal muscle

pathologies and could depend on whether the skel-

etal muscle damage is a single event, as in trauma,

IMMUNOCHEMICAL DETECTION OF HUMAN SKELETAL TnI 359

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

or a prolonged process characteristic, for example,

to dystrophies or muscle atrophy. Different compo-

sitions of troponin forms in blood could be present

in different diseases, and differential recognition of

these forms could serve as a diagnostic marker for

the detection of some pathological conditions in the

skeletal muscle.

In addition to differential detection of various

troponin forms, we developed FIAs that could detect

fsTnI and ssTnI individually and simultaneously. The

epitopes of mAbs that detect fsTnI (skTnI89-skTnI91)

and ssTnI (skTnI58-skTnI27) are specific to the central

regions of the proteins. These are the most variable

parts of the molecules, and utilization of the mAbs

specific to these regions facilitates development of

the methods that do not cross-react with other TnI

isoforms. In contrast, the mAbs for detection of both

skeletal TnI isoforms (skTnI25-skTnI50) are specific to

the C-terminal region of the proteins. Although this is

the most conserved part of the TnI isoform, it does

not cross-react with cTnI. FIAs that specifically detect

fsTnI or ssTnI could be used to diagnose diseases in

which muscle fibers of a particular type are selective-

ly damaged. This may be important for differential

diagnosis of certain pathologies because fast and slow

muscle fibers have different regeneration patterns

and require different therapeutic approaches [55]. An

assay that detects both skeletal TnI isoforms could be

used to detect general muscle damage.

Based on the similarity of skeletal TnI isoforms

with cTnI, it could be assumed that the skeletal TnI

is also present in the blood as part of ternary or bi-

nary complexes. Therefore, one of the goals of this

study was to develop immunochemical systems that

would effectively recognize all possible forms of TnI.

However, during the development of sandwich FIAs,

recognition of ITC and IC complexes in some assays

was significantly lower than that of the free TnI. To

achieve comparable recognition of free TnI and its

complexes, we adopted a previously established ap-

proach: addition of EDTA to the antibody dilution

buffer, which presumably leads to dissociation of

the troponin complex or changes in its conformation

so that the TnI epitopes are exposed for interaction

with antibodies [14,  21,  56]. In the presence of EDTA,

the sandwich FIAs that detect fsTnI and ssTnI recog-

nize not only free proteins, but also TnI as part of

the IC and ITC complexes. Necessity for EDTA addi-

tion leading to dissociation/conformational changes

is consistent with the epitope specificity of the mAbs

we obtained. In particular, according to the data

on the structure of troponins within the troponin

complex (Fig.  1), mAb skTnI89

86-105

(fsTnI) and mAb

skTn27

58-63

(ssTnI) recognize the site of interaction

with TnT, which was confirmed by the recovery of

immunochemical signals when EDTA was added to

the samples containing fsITC (Fig.5a, curves 5 and6)

and ssITC (Fig.  6). Accuracy of the epitope mapping

of skTnI91

30-49

(fsTnI) and skTnI58

30-49

(ssTnI) did not

allow us to determine whether these mAbs bind to

TnI at the interaction site with TnC. However, accord-

ing to the results obtained in this study, addition of

EDTA had no effect on immunochemical signal in the

samples containing fsIC (Fig. 5a, curves 3 and 4) and

ssIC (Fig.  5b, curves 3 and 4). Hence, we could as-

sume that the epitopes of these mAbs are not shielded

by TnC.

The results obtained for the skTnI25-skTnI50 as-

say could not be clearly interpreted. Immunochem-

ical activities of the binary (fsIC and ssIC) and ter-

nary (fsITC) complexes changed after EDTA addition

(Fig.  5). This is despite the fact that both epitopes of

the mAbs are specific to the C-terminal parts of the

molecules, which are not shielded by other troponins

(Fig.  1). We hypothesized that this could be due to

conformational changes that affect C-terminal region

of TnI when it interacts with TnC and TnT. However,

this requires further investigation.

Data on the basal concentrations of skeletal TnI

isoforms are controversial. Some studies did not de-

tect skeletal TnI in the healthy individuals [10,  14,

19,  56,  57], which could be associated with insuffi-

cient sensitivity of the assays used. In other studies,

concentrations of the skeletal TnI in the healthy vol-

unteers measured with the assay recognizing both

isoforms were: 1.74  ±  0.27  ng/mL, 0.5  ng/mL (inter-

quartile range, 0.3-0.9  ng/mL), and 2.5  ±  0.9  ng/mL

[11,  13,  15]. The reported data regarding troponin

levels under various conditions accompanied by

the skeletal muscle damage, provide the following

mean concentrations of the skeletal TnI: after high

intensity exercise – 62.2  ±  139  ng/mL [11], 6  h after

running downhill – 27.3  ng/mL (interquartile range,

8.5-43  ng/mL), 6  h after level running – 6.6  ng/mL

(3.7-9  ng/mL), and 24  h after eccentric contractions

of the quadriceps femoris muscle – 6.8  ng/mL (3.1-

14.9  ng/mL) [12]. The median concentrations of skel-

etal TnI isoforms obtained within 24 h after injury

were reported as 15.3  ±  2.4  ng/mL after orthopedic in-

jury and 10.4  ±  1.8  ng/mL after soft tissue injury [13].

Median concentration of the skeletal TnI isoforms

in the patients with inflammatory myopathies was

8.6  ng/mL (interquartile range, 3.2-33.5  ng/mL) [14].

These data are summarized in Table  S2 in the Online

Resource 1. LODs of the developed test systems were

in the range 0.07-0.11  ng/mL, and their linear ranges

were between 0.08-0.31 and 80-640  ng/mL (Table  1).

Considering the aforementioned data, sensitivity and

linear range of the sandwich FIAs designed in this

study are sufficient for reliable quantitative detection

of the skeletal TnI isoforms in blood samples of the

patients with skeletal muscle injuries.

BOGOMOLOVA et al.360

BIOCHEMISTRY (Moscow) Vol. 90 No. 3 2025

CONCLUSION

The present study aimed to develop different

methods for specific immunochemical detection of the

human skeletal TnI isoforms for various applications,

including WB, IHC staining, and FIA.

We developed specific and sensitive methods

for detection of fast and slow skeletal TnI isoforms

(together and separately) by WB. Our mAbs also rec-

ognize the skeletal TnI isoforms in human tissues by

IHC, differentiated slow and fast fibers, and detected

slow skeletal isoforms in the nondifferentiated cardio-

myocytes.

Considering that the skeletal TnI isoforms are

potential markers of the skeletal muscle damage of

various etiologies, we developed methods based on

FIA to determine concentrations of the fast and slow

isoforms separately and together. Sensitivity of these

assays is sufficient to differentiate healthy individuals

from those with skeletal muscle injuries. To date, it is

still unclear whether the skeletal TnI isoforms are re-

leased into the human bloodstream as free proteins or

in complexes with TnC and TnT. Several assays have

been designed to detect skeletal TnI in human blood,

both in the free form and as part of binary and ter-

nary troponin complexes.

In addition, we designed immunoassays that

could differentially detect various forms of skeletal

TnI isoforms, including binary IC complexes with TnC

or ternary ITC complexes with TnT and TnC. These

methods could facilitate investigation of the presence

of different forms of skeletal TnI isoforms in the hu-

man bloodstream.

Abbreviations. Anti-MyHC, antibodies specific to

MHC; cIC, cardiac IC; DAB, diaminobenzidine; ELISA,

enzyme-linked immunosorbent assay; FIA, fluores-

cence immunoanalysis; fsTnI, fast skeletal troponinI;

fsIC, fast skeletal IC; fsITC, fast skeletal ITC; IC, binary

TnI–TnC complex; ITC, ternary TnI–TnT–TnC complex;

LOD, limit of detection; mAb, monoclonal antibody;

PBS, phosphate buffered saline; PBST, PBS contain-

ing 0.1% Tween-20; SD, standard deviation; ss/cTnC,

slow skeletal/cardiac troponin C; ssIC, slow skeletal IC;

ssITC, slow skeletal ITC; ssTnI, slow skeletal troponinI;

TnC, troponin C; TnI, troponin I; TnT, troponin T; WB,

western blotting.

Supplementary information. The online version

contains supplementary material available at https://

doi.org/10.1134/S0006297924601928.

Contributions. I. A. Katrukha and A. G. Katrukha

conceived and supervised the study; A. P. Bogomolo-

va, A. V. Bereznikova, A. M. Emelin, R. V. Deev, and

A. I. Zabolotsky carried out experiments; A. P. Bogo-

molova, I. A. Katrukha, and O. S. Lebedeva discussed

the results of experiments with input from all authors;

A. P. Bogomolova, I. A. Katrukha, A. M. Emelin, and

R. V. Deev wrote the manuscript; I. A. Katrukha,

A. P. Bogomolova, and O. S. Lebedeva edited the man-

uscript.

Funding. This work was financially supported

by Hytest (Moscow, Russia) and Hytest Ltd. (Turku,

Finland).

Ethics approval and consent to participate.

This study was conducted in accordance with the

current version of the Declaration of Helsinki. All

samples and tissues were collected according to a

protocol approved by the local Ethical Committee at

the Lopukhin Federal Research and Clinical Center

of Physical-Chemical Medicine at the Federal Medi-

cal Biological Agency of Russia on February 06, 2024

(meeting number, 2024/02/06).

Conflict of interest. The authors declare no con-

flicts of interest.

Open access. This article is licensed under a Cre-

ative Commons Attribution 4.0 International License,

which permits use, sharing, adaptation, distribution,

and reproduction in any medium or format, as long

as you give appropriate credit to the original author(s)

and the source, provide a link to the Creative Com-

mons license, and indicate if changes were made.

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are included in the article’s Creative Commons license,

unless indicated otherwise in a credit line to the mate-

rial. If material is not included in the article’s Creative

Commons license and your intended use is not permit-

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use, you will need to obtain permission directly from

the copyright holder. To view a copy of this license,

visit http://creativecommons.org/licenses/by/4.0/.

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