Generation and characterization of monoclonal antibodies specific to Coenzyme A
im. Generation of monoclonal antibodies specific to Coenzyme A. Methods. Hybridoma technique. KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifically recognizes CoA and Co...
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| Опубліковано в: : | Вiopolymers and Cell |
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| Дата: | 2015 |
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| Формат: | Стаття |
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Інститут молекулярної біології і генетики НАН України
2015
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Generation and characterization of monoclonal antibodies specific to Coenzyme A / O.M. Malanchuk, G.G. Panasyuk, N.M. Serbyn, I.T. Gout, V.V. Filonenko // Вiopolymers and Cell. — 2015. — Т. 31, № 2. — С. 187-192. — Бібліогр.: 17 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859669200891543552 |
|---|---|
| author | Malanchuk, O.M. Panasyuk, G.G. Serbyn, N.M. Gout, I.T. Filonenko, V.V. |
| author_facet | Malanchuk, O.M. Panasyuk, G.G. Serbyn, N.M. Gout, I.T. Filonenko, V.V. |
| citation_txt | Generation and characterization of monoclonal antibodies specific to Coenzyme A / O.M. Malanchuk, G.G. Panasyuk, N.M. Serbyn, I.T. Gout, V.V. Filonenko // Вiopolymers and Cell. — 2015. — Т. 31, № 2. — С. 187-192. — Бібліогр.: 17 назв. — англ. |
| collection | DSpace DC |
| container_title | Вiopolymers and Cell |
| description | im. Generation of monoclonal antibodies specific to Coenzyme A. Methods. Hybridoma technique. KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifically recognizes CoA and CoA derivatives, but not its precursors ATP and cysteine has been generated. Conclusion. In this study, we describe for the first time the production and characterization of monoclonal antibodies against CoA. The monoclonal antibody 1F10 was shown to recognize specifically CoA in Western blotting, ELISA and immunoprecipitation. These properties make this antiboby a particularly valuable reagent for elucidating CoA function in health and disease.
Ціль. Отримати та охарактеризувати моноклональні антитіла, специфічні до КоА. Методи. Гібридомна технологія. Для імунізації було використано КоА, кон’югований з білком-носієм KLH. Скринінг позитивних клонів проводили з використанням БСА, кон’югованого з КоА. Результати. Отримано моноклональні антитіла, що специфічно розпізнають КоА та КоА-похідні та не розпізнають його попередників – АТФ та цистеїну. Висновки. Вперше описано створення та характеристику моноклональних антитіл проти КоА. Моноклональні антитіла 1F10 специфічно розпізнають КоА в Вестерн блотингу, ІФА та імунопреципітації. Такі властивості антитіл вказують на перспективність використання для аналізу функцій КоА в нормі та за патологій.
Цель. Получить и охарактеризовать моноклональные антитела, специфичные к КоА. Методы. Гибридомная технология. Для иммунизации был использован КоА, конъюгированный с белком-носителем KLH. Скрининг положительных клонов проводили с использованием БСА, конъюгиванного с КоА. Результаты. Получено моноклональные антитела, которые специфично распознают КоА и КоА-производные и не распознают его предшественников – АТФ и цистеина. Выводы. Впервые описано получение моноклональных антител к КоА. Показано, что моноклональные антитела 1F10 специфически распознают КоА в вестерн плоте, ИФА и иммунопреципитации. Такие свойства антител указывают на возможность их использования для анализа функций КоА в норме и при патологиях.
|
| first_indexed | 2025-11-30T13:00:52Z |
| format | Article |
| fulltext |
187
ISSN 0233-7657
Biopolymers and Cell. 2015. Vol. 31. N 3. P. 187–192
doi: http://dx.doi.org/10.7124/bc.0008DF
UDC 576.322 577.22
Generation and characterization of monoclonal
antibodies specifi c to Coenzyme A
O. M. Malanchuk1, G. G. Panasyuk1, N. M. Serbin1, I. T. Gout2, V. V. Filonenko1
1 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2 Institute of Structural and Molecular Biology, University College London,
Gower Street, London, WC1E 6BT, UK
fi lonenko@imbg.org.ua
Aim. Generation of monoclonal antibodies specifi c to Coenzyme A. Methods. Hybridoma technique.
KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was
performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifi cally recognizes CoA
and CoA derivatives, but not its precursors ATP and cysteine has been generated. Conclusion. In this
study, we describe for the fi rst time the production and characterization of monoclonal antibodies against
CoA. The monoclonal antibody 1F10 was shown to recognize specifi cally CoA in Western blotting,
ELISA and immunoprecipitation. These properties make this antiboby a particularly valuable reagent for
elucidating CoA function in health and disease.
K e y w o r ds: CoA, hybridoma technique, monoclonal antibody.
© 2015 O. M. Malanchuk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
Introduction
Coenzyme A (CoA) is an essential cofactor in all liv-
ing organisms. CoA and its thioester derivatives (ace-
tyl-CoA, malonyl-CoA, HMG-CoA etc) participate
in the diverse anabolic and catabolic pathways. Apart
from participating in cellular metabolism as sub-
strates and intermediates, CoA and its derivatives,
such as acetyl-CoA, can also directly regulate the ac-
tivity of proteins by allosteric mechanisms and ge ne
expression by protein acetylation [1].
The levels of CoA and its derivatives in mamma-
lian cells and tissues as well as in different cellular
compartments are tightly regulated by a diverse ran-
ge of stimuli and stresses, including nutrients, hor-
mones, cellular metabolites, fasting and refeeding. It
has been demonstrated that the total level of CoA is
reduced in response to insulin, glucose, fatty acids
and pyruvate, whereas glucagon and glucocorticoids
have an opposite effect [2, 3]. The changes in the le-
vel of CoA occur at several pathological conditions,
such as diabetes, cancer and cardiac hypertrophy [4–
12]. Defective CoA biosynthesis is implicated in neu-
rodegeneration with brain iron accumulation (NBIA)
[13–15]. However, the exact role of CoA and its de-
rivatives in the pathogenesis of the above disorders
is not well understood. The reliable and accurate as-
says for measuring CoA species in biological sam-
ples are essential for better understanding of the
roles of CoA and CoA derivatives under physiologi-
cal and pathological conditions.
At present, there are a number of methods for meas-
uring CoA and its derivatives based on enzymatic as-
says, HPLC and mass spectrometry quantifi cations
[16]. However, each of these approaches has its own
limitations. Therefore, development of novel tools and
approaches for measuring CoA species in different sub-
cellular compartments or different cell types and tissues
would be highly useful for studying the consequences
of altered CoA levels on cellular processes.
188
O. M. Malanchuk, G. G. Panasyuk, N. M. Serbin, I. T. Gout, V. V. Filonenko
The aim of this work was to generate monoclonal
antibodies specifi c to CoA which could be used in
research and for the development of highly sensitive
ELISA-based assays aimed at detecting CoA and its
derivatives in biological samples.
Materials and Methods
Generation of antigens for mice immunization, scree-
ning of hybridoma clones and antibodies characteri-
sation. For mice immunization and selection of pos-
itive clones, CoA was directly conjugated to maleimi-
de-activated keyhole limpet hemocyanin (KLH) and
bovine serum albumin (BSA) correspondingly. In bri-
ef, 1 mg of maleimide-activated KLH or BSA (Pier-
ce) was dissolved in 500 ul H2O and then mixed with
500 μl of CoA (0,5 mg/ml in H2O). The mix was in-
cubated on the wheel at room temperature for 2hrs be-
fore extensive dialysis in PBS. After testing the effi -
ciency of conjugation, prepared samples of KLH-CoA
and BSA-CoA were aliquoted and stored at –20 C.
To test the specifi city of anti-CoA antibodies in
WB and immunoprecipitation assays CoA was re-
versibly cross-linked to BSA with the use of N-suc-
cinimidyl 3-(2-pyridyldithio) propionate (SPDP, Pier-
ce) according to the manufacturer’s recommenda-
tions. For this, BSA (3 mg) in 100 ul PBS was mixed
with SPDP (0.36 mg) dissolved in 10 μl of DMSO
and incubated at 37 C for 1h. Activated BSA was
desalted on G25 column to remove unreacted SPDP
and incubated for 2h at 37 C with 0.2 mg CoA in a
total volume of 11 0μl. Unreacted CoA was removed
by desalting on G25 column and BSA-S-CoA was
aliqouted and stored at –20 C.
Production of hybridomas. Female BALB/c mice
(6–8 weeks old) were immunized with 20 μg of
KLH-CoA in complete Freund’s adjuvant by intra-
peritoneal injections (i.p.) every two weeks. When
the titer of anti-CoA antibody in the sera of immu-
nized mice reached 10–4, the production of hybrido-
mas was performed according to a standard protocol
[17]. In brief, immunized mice were boosted with 20
μg of antigen in PBS by i.p. injection. Three days
la ter, spleenocytes from immunized mice and SP2/0
myeloma cells cultured in RPMI 1640 medium con-
taining 20 % fetal calf serum (FCS) were fused in
the presence of PEG (MW2000, Merck, Germany).
Primary screening of hybridoma culture media was
performed 7 days later using ELISA. Isolated posi-
tive clones were subcloned by limiting dilution me-
thod [17].
ELISA assay. Ninety-six-well polystyrene plates
were incubated with 0.3 μg/well of BSA-CoA for 2
Ac etyl
C oA
C oenzyme A
(C oA)
S uc c inyl
C oA
F atty
ac yl C oA
Malonyl
C oAHMG
C oA
P ropion
yl C oA
B utyryl
C oA
Fig. 1. Schematic structure of CoA and its thioester derivatives
189
Generation and characterization of monoclonal antibodies specifi c to Coenzyme A
h at 37 °C. The plates were then washed three times
with PBS containing 0.1 % Tween-20 (PBS-T) (Sig-
ma, USA) and incubated with 200 μl of 2 % bovine
serum albumin (BSA) in PBS (pH 7.4) for 1h at ro-
om temperature in order to block non-specifi c bind-
ing of antibodies. Subsequently, the plates were
loaded with 100 μl aliquots of hybridoma media and
were incubated for 1 h at 37 °C followed by three
washes with PBS-T. After washing, 100 μl of horse-
radish peroxidase (HRP)-conjugated goat anti-mouse
IgG antibodies (1:5000 v/v, Promega, USA) were
added to each well and incubated at 37 °C for 1 h.
Plates were washed again three times, before 100 μl
of substrate solution (0.02 % H2O2, 0.5 mg/ml 2.2’-
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
sodium salt (Sigma, USA), 0.1 M citrate-phosphate
buffer (pH 5.8)) was added to each well. After 15-
min incubation at 37 °C, the absorbance of the each
well was determined at 490 nm.
Competitive ELISA. This analysis was perfor med
according to the above protocol with minor modifi -
cations. The assay was carried out in 96 well plates,
containing immobilized BSA-CoA (0.3 μg/well). The
primary mAb 1F10 (10 μg in 100 ul PBS) was then
added together with different concentrations (5
10–1 mM, 5 10–2 mM, 5 10–3 mM, 5 10–4 mM,
5 10–5 mM, 5 10–6 mM) of CoA, ATP, cysteine,
succinyl-CoA, malonyl-CoA or glutaryl-CoA. The
mixture was incubated for 1hr at room temperature
and the assay developed as described above.
Western blot analysis. BSA-S-CoA (100 ng per well)
was heat treated in sample buffer with and without
DTT, resolved by SDS/PAGE and electrotransferred
onto Immobilon-P membrane (Millipore, USA). The
membrane was divided into strips and blocked with
0.5 % BSA in PBS for 1 h at room temperature fol-
lowed by a single wash with PBS containing 0.1 %
Tween 20. Strips were incubated with hybridoma
media from selected clones for 2 h at room tempera-
ture. After three washes, a peroxidase-conjugated anti-
mouse secondary antibody (Promega, USA) was ad-
ded to the strips and incubated for 1 h at room tem-
perature. Strips were washed three times, and devel-
oped by ECL detection system (Amersham, Sweden).
Immunoprecipitation. Culture media (500 μl) from
selected positive hybridoma clones 1F10/B11, 9F10/
B6 and 9F6/G1 were incubated with 25 μl of 50 %
suspension of Protein A Sepharose CL-4B (Amer sham,
Fig. 2. (A) Schematic structures of KLH-CoA and BSA-CoA conjugates used for immunization and hybridoma screening respectively.
(B) Gel electrophoresis of BSA cross-linked with CoA by SPDP. BSA-S-CoA (2 μg) untreated (1) and treated (2) with DTT. 3 –
protein standards. SDS-PAGE (10 %)
A B
BSA-S-CoA
100
70
130
55
Mr, kDa
170
– DTT + DTT
1 2 3
BSA-S-CoA
100
70
130
55
Mr, kDa
170
– DTT + DTT
1 2 3
BSA-S-CoA
BSA-CoA
BSA
BSA-CoA
BSA
CoA
CoA
KLH-CoA
KLH
CoA
KLH-CoA
KLH
190
O. M. Malanchuk, G. G. Panasyuk, N. M. Serbin, I. T. Gout, V. V. Filonenko
Sweden) on the wheel for 2 h at 4 °C. Then be ads were
washed twice with PBS and incubated with 0,5 μg
BSA-S-CoA in PBS (500 μl) for 2 h at 4 °C. After incu-
bation, beads were washed three times with 1 ml PBS
containing 0,1 % Triton X-100. Im mune complexes
were removed from beads by boiling in Laemmli sam-
ple buffer (with or without DTT), separated by SDS-
PAGE and immunoblotted with mAbs 1F10/B11.
Purifi cation of MAbs from ascitic fl uid. BALB/c
mice were injected with 0.5 ml of Pristane and 7–10
days later inoculated with 5 · 106 of hybridoma cells.
The ascitic fl uid was collected after 7–10 days. The
fraction of immunoglobulins was precipitated from
ascitic fl uid with 50 % ammonium sulfate and used
for affi nity purifi cation by Protein A Sepharose CL-
4B (Amersham, Sweden) chromatography. The IgG
fractions were pulled together and dialyzed in a PBS.
The aliquots of purifi ed antibodies were stored at
–70 °C with 50 % glycerol.
Results and Discussion
Coenzyme A was discovered by Lipmann in 1945 as a
heat-stable cofactor required for many enzyme-cata-
lysed acetylation reactions. He later determined the
structure for CoA and was awarded the Nobel Prize in
1953 for this achievement. CoA has a unique structure
which allows it to function as a master acyl group car-
rier and carbonyl-activating group, resulting in a diverse
range of metabolically active thioester derivatives, in-
cluding acetyl-CoA, malonyl-CoA, 3-hyd roxy-3-me t-
hy lglutaryl-CoA etc (Fig. 1). In spite of a vast interest in
elucidating the regulation and function of CoA and its
derivatives under physiological and pathological condi-
tions, there are no commercially available polyclonal or
monoclonal antibodies specifi c to CoA.
The main focus of this study was to generate mon-
oclonal antibodies against CoA, which could be used
in various immunological assays, including Western
blotting, immunoprecipitation, and ELISA to allow a
more detailed study of the physiological and patho-
physiological role of CoA in health and disease. In
order to obtain monoclonal anti-CoA antibodies, we
prepared CoA conjugated to maleimide-activated
KLH and BSA (Fig. 2). Immunization of animals
was performed according to the standard protocol
using KLH-CoA as an antigen. The titer of anti-CoA
antibodies in immunized mice was monitored using
BSA-CoA. When the titer of anti-CoA antibody in
the sera of immunized mice reached 10–4, spleen
cells were fused with the Sp2/0 myeloma cells using
the PEG method according to [17]. Screening of
positive anti-CoA hybridoma clones was performed
against BSA-CoA conjugate. Following two rounds
1F
10
/B
11
1F
10
/F
7
9F
6/
G
1
9F
10
/B
6
c-medium
+DTT-DTT
1F
10
/B
11
1F
10
/F
7
9F
6/
G
1
9F
10
/B
6
WB:
70
130
55
100
BSA-S-CoA
M, kDa
Fig. 3. Testing the specifi city of anti-CoA mAbs. WB ana lysis of
BSA-S-CoA (100ng) by anti-CoA mAbs of hyb ri doma clones
1F10, 9F6 and 9F10. BSA-S-CoA samples be fo re electroforetical
separation were treated in sample buffer with or without DTT
Fig. 4. Testing of selected anti-CoA mAbs in immunoprecipitation as-
say. WB analysis of BSA-S-CoA immunoprecipitated by 1F10
mAbs. Immune complexes before electroforetical separation were
treated in sample buffer with or without DTT
BSA-S-CoA
IgG HCh
-DTT +DTT
B
SA
-S
-C
oA
+
Se
ph
B
SA
-S
-C
oA
+1
F1
0
+
Se
ph
1F
10
+
S
ep
h
B
SA
-S
-C
oA
+
Se
ph
1F
10
+
S
ep
h
B
SA
-S
-C
oA
+1
F1
0
+
Se
ph
IP:
WB: 1F10
IgG
70
130
55
100
M, kDa
BSA-S-CoA
IgG HCh
-DTT +DTT
B
SA
-S
-C
oA
+
Se
ph
B
SA
-S
-C
oA
+1
F1
0
+
Se
ph
1F
10
+
S
ep
h
B
SA
-S
-C
oA
+
Se
ph
1F
10
+
S
ep
h
B
SA
-S
-C
oA
+1
F1
0
+
Se
ph
IP:
WB: 1F10
IgG
70
130
55
100
M, kDa
191
Generation and characterization of monoclonal antibodies specifi c to Coenzyme A
of ELISA screening, only three positive hybridoma
clones were selected and designated as 9F6, 9F10 and
1F10. To confi rm the specifi city of selected hybridoma
clones against CoA, we analyzed their specifi city to-
wards CoA reversibly crosslinked to BSA (Fig. 2B) by
SPDP. SPDP is a short-chain crosslinker for amine-to-
sulfhydryl conjugation via NHS-ester and pyridyldithi-
ol reactive groups that form cleavable (reducible) di-
sulfi de bonds with cysteine sulfhydryls. As shown in
Fig. 3, only subclones of 1F10 hybridoma showed spe-
cifi c recognition of CoA conjugated with BSA. When
BSA-S-CoA was treated with DTT, 1F10 mAbs could
not recognize the antigen demonstrating the specifi city
towards CoA (Fig. 3). Then, we tested the ability of
selected hybrid clones to immunoprecipitate BSA-S-
CoA conjugate. This analysis revealed that only 1F10
mAb specifi cally precipitated BSA conjugated with
CoA (Fig. 4). However, the immunoprecipitation of
BSA-S-CoA was not very effi cient, indicating low af-
fi nity of 1F10 mAb towards CoA. No specifi c immu-
noprecipitation of BSA-S-CoA under various experi-
mental conditions was observed with hybridoma clones
9F6 and 9F10 (data not shown).
The biosynthesis of CoA involves fi ve enzymatic
steps that are highly conserved from prokaryotes to eu-
karyotes and requires pantothenic acid, cysteine and
ATP. (Fig. 5). Hypothetically, the 1F10 epitope may
correspond to various regions of CoA structure. We
have attempted to locate the epitope for 1F10 mAb by
performing competitive ELISA (Fig. 5) and demon-
strated that ATP and cysteine did not compete with CoA
for the interaction with anti-CoA mAb (1F10). At the
same time, CoA and its derivatives succinyl-CoA, mal-
onyl-CoA and glutaryl-CoA effi ciently deprived the
recognition of BSA-CoA by 1F10 mAb. Further stud-
ies showed that the 1F10 epitope doesn’t correspond to
the panthothenate moiety of CoA (data not shown).
Taking this into account, data suggest that the antigenic
determinant for 1F10 mAbs may cover the junctions
between ATP and pantothenate or pantothenate and
cysteine. We can’t exclude that the 1F10 epitope covers
all three blocks of CoA. Further studies are required to
map the 1F10 epitope with more precision.
In conclusion, this study describes the produc-
tion and characterisation of fi rst monoclonal anti-
body that specifi cally recognises CoA and its de-
rivatives. We anticipate that generated anti-CoA
1F10 antibody could be used for academic re-
search and the development of reliable and highly
sensitive ELISA-based immunoassays, allowing
the detection of CoA and its derivatives in biolog-
ical samples.
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E 1
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Отримання та характеристика
моноклональних антитіл, специфічних до КоА
О. М. Маланчук, Г. Г. Панасюк,
Н. М. Сербин, І. Т. Гут, В. В. Філоненко
Мета. Отримати та охарактеризувати моноклональні антиті-
ла, специфічні до КоА. Методи. Гібридомна технологія. Для
імунізації було використано КоА, кон’югований з білком-
носієм KLH. Скринінг позитивних клонів проводили з ви-
користанням БСА, кон’югованого з КоА. Результати. От ри-
мано моноклональні антитіла, що специфічно розпізнають
КоА та КоА-похідні та не розпізнають його попередників –
АТФ та цистеїну. Висновки. Вперше описано створення та
характеристику моноклональних антитіл проти КоА. Моно-
клональні антитіла 1F10 специфічно розпізнають КоА в
Вестерн блотингу, ІФА та імунопреципітації. Такі властивос-
ті антитіл вказують на перспективність ви користання для ана-
лізу функцій КоА в нормі та за патологій.
Ключов і слова: КоА, гібридомна технологія, мо но кло-
на ль ні ан титіла.
Получение и характеристика
моноклональных антител, специфичных к КоА
О. Н. Маланчук, А. Г. Панасюк,
Н. Н. Сербин, И. Т. Гут, В. В. Филоненко
Цель. Получить и охарактеризовать моноклональные анти-
тела, специфичные к КоА. Методы. Гибридомная техноло-
гия. Для иммунизации был использован КоА, конъюгиро-
ванный с белком-носителем KLH. Скрининг положительных
клонов проводили с использованием БСА, конъюгиванного
с КоА. Результаты. Получено моноклональные антитела,
которые специфично распознают КоА и КоА-производные и
не распознают его предшественников – АТФ и цистеина.
Выводы. Впервые описано получение моноклональных ан-
тител к КоА. Показано, что моноклональные антитела 1F10
специфически распознают КоА в вестерн плоте, ИФА и им-
мунопреципитации. Такие свойства антител указывают на
возможность их использования для анализа функций КоА в
норме и при патологиях.
Ключевые слова: КоА, гибридомная технология, мо но-
кло нальные антитела.
Received 28.03.2015
|
| id | nasplib_isofts_kiev_ua-123456789-152437 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-11-30T13:00:52Z |
| publishDate | 2015 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Malanchuk, O.M. Panasyuk, G.G. Serbyn, N.M. Gout, I.T. Filonenko, V.V. 2019-06-11T17:21:37Z 2019-06-11T17:21:37Z 2015 Generation and characterization of monoclonal antibodies specific to Coenzyme A / O.M. Malanchuk, G.G. Panasyuk, N.M. Serbyn, I.T. Gout, V.V. Filonenko // Вiopolymers and Cell. — 2015. — Т. 31, № 2. — С. 187-192. — Бібліогр.: 17 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008DF https://nasplib.isofts.kiev.ua/handle/123456789/152437 576.322 577.22 im. Generation of monoclonal antibodies specific to Coenzyme A. Methods. Hybridoma technique. KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifically recognizes CoA and CoA derivatives, but not its precursors ATP and cysteine has been generated. Conclusion. In this study, we describe for the first time the production and characterization of monoclonal antibodies against CoA. The monoclonal antibody 1F10 was shown to recognize specifically CoA in Western blotting, ELISA and immunoprecipitation. These properties make this antiboby a particularly valuable reagent for elucidating CoA function in health and disease. Ціль. Отримати та охарактеризувати моноклональні антитіла, специфічні до КоА. Методи. Гібридомна технологія. Для імунізації було використано КоА, кон’югований з білком-носієм KLH. Скринінг позитивних клонів проводили з використанням БСА, кон’югованого з КоА. Результати. Отримано моноклональні антитіла, що специфічно розпізнають КоА та КоА-похідні та не розпізнають його попередників – АТФ та цистеїну. Висновки. Вперше описано створення та характеристику моноклональних антитіл проти КоА. Моноклональні антитіла 1F10 специфічно розпізнають КоА в Вестерн блотингу, ІФА та імунопреципітації. Такі властивості антитіл вказують на перспективність використання для аналізу функцій КоА в нормі та за патологій. Цель. Получить и охарактеризовать моноклональные антитела, специфичные к КоА. Методы. Гибридомная технология. Для иммунизации был использован КоА, конъюгированный с белком-носителем KLH. Скрининг положительных клонов проводили с использованием БСА, конъюгиванного с КоА. Результаты. Получено моноклональные антитела, которые специфично распознают КоА и КоА-производные и не распознают его предшественников – АТФ и цистеина. Выводы. Впервые описано получение моноклональных антител к КоА. Показано, что моноклональные антитела 1F10 специфически распознают КоА в вестерн плоте, ИФА и иммунопреципитации. Такие свойства антител указывают на возможность их использования для анализа функций КоА в норме и при патологиях. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers Generation and characterization of monoclonal antibodies specific to Coenzyme A Отримання та характеристика моноклональних антитіл, специфічних до КоА Получение и характеристика моноклональных антител, специфичных к КоА Article published earlier |
| spellingShingle | Generation and characterization of monoclonal antibodies specific to Coenzyme A Malanchuk, O.M. Panasyuk, G.G. Serbyn, N.M. Gout, I.T. Filonenko, V.V. Structure and Function of Biopolymers |
| title | Generation and characterization of monoclonal antibodies specific to Coenzyme A |
| title_alt | Отримання та характеристика моноклональних антитіл, специфічних до КоА Получение и характеристика моноклональных антител, специфичных к КоА |
| title_full | Generation and characterization of monoclonal antibodies specific to Coenzyme A |
| title_fullStr | Generation and characterization of monoclonal antibodies specific to Coenzyme A |
| title_full_unstemmed | Generation and characterization of monoclonal antibodies specific to Coenzyme A |
| title_short | Generation and characterization of monoclonal antibodies specific to Coenzyme A |
| title_sort | generation and characterization of monoclonal antibodies specific to coenzyme a |
| topic | Structure and Function of Biopolymers |
| topic_facet | Structure and Function of Biopolymers |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/152437 |
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