Immunological images of polycyclic aromatic hydrocarbons
Aim: To develop experimental model for definition of immunological images of chemical carcinogens. Materials and Methods: The conjugates of benzo[a]pyrene, benz[a]anthracene, anthracene, chrysene and pyrene with bovine serum albumin and yeast hexokinase were synthesized. Rabbits were immunized by bo...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2006
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| Назва видання: | Experimental Oncology |
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| Цитувати: | Immunological images of polycyclic aromatic hydrocarbons / A.N. Glushkov, M.V. Kostyanko, T.P. Anosova, E.G. Polenok, S.A. Mun, M.P. Anosov, S.V. Cherno // Experimental Oncology. — 2006. — Т. 28, № 2. — С. 166-168. — Бібліогр.: 8 назв. — англ. |
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irk-123456789-1375652018-06-18T03:07:33Z Immunological images of polycyclic aromatic hydrocarbons Glushkov, A.N. Kostyanko, M.V. Anosova, T.P. Polenok, E.G. Mun, S.A. Anosov, M.P. Cherno, S.V. Short communications Aim: To develop experimental model for definition of immunological images of chemical carcinogens. Materials and Methods: The conjugates of benzo[a]pyrene, benz[a]anthracene, anthracene, chrysene and pyrene with bovine serum albumin and yeast hexokinase were synthesized. Rabbits were immunized by bovine serum albumin-hapten conjugates. Antibodies to each hapten were isolated from the serum by affinity chromatography with the hapten-yeast hexokinase-Sepharose sorbents. The binding of each hapten with each antibody was determined by competitive immunoassay. Results: The immunological images of all the investigated chemical compounds were described. Conclusion: The model is proposed to determine the internal immunological images of anti-idiotypic monoclonal antibodies to chemical carcinogens. Цель: разработка экспериментальной модели для определения иммунологических образов химических канцерогенов. Материалы и методы: синтезированы конъюгаты бензо[a]пирена, бенз[a]антрацена, антрацена, пирена и хризена с бычьим сывороточным альбумином и дрожжевой гексокиназой. Кроликов иммунизировали конъюгатами с бычьим сывороточным альбумином в качестве белка-носителя. Антитела к каждому из гаптенов выделяли из сыворотки аффинной хроматографией на сорбентах гаптен-дрожжевая гексокиназа-Sepharose. Способность антител связываться с соответствующими гаптенами определяли с помощью конкурентного иммуноанализа. Результаты: в результате исследования охарактеризованы иммунологические образы исследуемых химических канцерогенов. Выводы: предложенная модель дает возможность определять внутренние иммунологические образы антиидиотипических моноклональных антител к химическим канцерогенам. 2006 Article Immunological images of polycyclic aromatic hydrocarbons / A.N. Glushkov, M.V. Kostyanko, T.P. Anosova, E.G. Polenok, S.A. Mun, M.P. Anosov, S.V. Cherno // Experimental Oncology. — 2006. — Т. 28, № 2. — С. 166-168. — Бібліогр.: 8 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137565 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Short communications Short communications |
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Short communications Short communications Glushkov, A.N. Kostyanko, M.V. Anosova, T.P. Polenok, E.G. Mun, S.A. Anosov, M.P. Cherno, S.V. Immunological images of polycyclic aromatic hydrocarbons Experimental Oncology |
| description |
Aim: To develop experimental model for definition of immunological images of chemical carcinogens. Materials and Methods: The conjugates of benzo[a]pyrene, benz[a]anthracene, anthracene, chrysene and pyrene with bovine serum albumin and yeast hexokinase were synthesized. Rabbits were immunized by bovine serum albumin-hapten conjugates. Antibodies to each hapten were isolated from the serum by affinity chromatography with the hapten-yeast hexokinase-Sepharose sorbents. The binding of each hapten with each antibody was determined by competitive immunoassay. Results: The immunological images of all the investigated chemical compounds were described. Conclusion: The model is proposed to determine the internal immunological images of anti-idiotypic monoclonal antibodies to chemical carcinogens. |
| format |
Article |
| author |
Glushkov, A.N. Kostyanko, M.V. Anosova, T.P. Polenok, E.G. Mun, S.A. Anosov, M.P. Cherno, S.V. |
| author_facet |
Glushkov, A.N. Kostyanko, M.V. Anosova, T.P. Polenok, E.G. Mun, S.A. Anosov, M.P. Cherno, S.V. |
| author_sort |
Glushkov, A.N. |
| title |
Immunological images of polycyclic aromatic hydrocarbons |
| title_short |
Immunological images of polycyclic aromatic hydrocarbons |
| title_full |
Immunological images of polycyclic aromatic hydrocarbons |
| title_fullStr |
Immunological images of polycyclic aromatic hydrocarbons |
| title_full_unstemmed |
Immunological images of polycyclic aromatic hydrocarbons |
| title_sort |
immunological images of polycyclic aromatic hydrocarbons |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2006 |
| topic_facet |
Short communications |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/137565 |
| citation_txt |
Immunological images of polycyclic aromatic hydrocarbons / A.N. Glushkov, M.V. Kostyanko, T.P. Anosova, E.G. Polenok, S.A. Mun, M.P. Anosov, S.V. Cherno // Experimental Oncology. — 2006. — Т. 28, № 2. — С. 166-168. — Бібліогр.: 8 назв. — англ. |
| series |
Experimental Oncology |
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| first_indexed |
2025-07-10T02:35:32Z |
| last_indexed |
2025-07-10T02:35:32Z |
| _version_ |
1837225669566660608 |
| fulltext |
166 Experimental Oncology 28, 166–168, 2006 (June)
New approaches for immunoprevention of cancer
and other diseases, induced by chemical carcinogens,
were established during last years [3]. The most ef-
fective vaccine against chemical carcinogens could
be anti-idiotypic monoclonal antibodies (mAb). They
are not carcinogenic and at the same time they carry
so called “internal immunological image” of a car-
cinogen and are able to induce anti-carcinogenic
Ab. Chagnaud et al. [1, 2] showed that anti-idiotypic
mAb to benzo[a]pyrene (Bp) slowed the appearance
and growth of tumors induced by Bp in experimental
animals. However, this method for preparation of mAb
does not specify which polycyclic aromatic hydrocar-
bon (PAH) corresponds to the internal immunological
image in Ab. The object of the present study is to
develop experimental model to define immunological
images of chemical carcinogens and to describe the
immunological images of Bp, benz[a]anthracene (Ba),
anthracene (Ac), chrysene (Cr) and pyrene (P).
Synthesis of PAH-protein conjugates. The
conjugates of Bp, Ba, Ac, Cr and P with bovine serum
albumin (BSA) for animal immunization and yeast
hexokinase (HK) for affinity immobilization were syn-
thesized by method described earlier [4, 5].
Five PAH-BSA immunogenes were synthesized
as followed. We mixed 1 ml BSA (100 mg dissolved in
0.1 M NaOH) and 1 ml pyridine with one of each com-
pound:17.5 mg Вр-6-; 14.6 mg Ва-7-; 11.7 mg Ас-9-;
16.0 mg Cr-6-; 10.2 mg Р-1-carboxaldehyde, diluted in
1.5 ml of pyridine. After 5 h of stirring, solution of 7.5 mg
sodium borohydride in 0.1 ml water was added. Then
after 1.5 h the excess of the reductant was decomposed
by 50 μl of acetic acid and protein was precipitated with
10 ml acetone. The precipitate was repeatedly washed
with acetone and immediately dried under vacuum.
The РАН-НК conjugates were synthesized from
70 mg of НК, 1 ml of 0.06 М NaOH and one of
7.3 mg Вр-6-; 7.9 mg Ва-7-; 6.0 mg Ас-9-; 8.5 mg Cr-6-
or 6.1 mg Р-1-carboxaldehyde by means of the above
method. 50 mg acrylamide was added at the stage of
reduction of the conjugates with sodium borohydride.
Hapten density for the obtained conjugates was
25 ± 2 and 15 ± 1 molecules of РАН per BSA and HK
molecules, respectively.
Animal immunisation. Rabbits were immunized
according to the following scheme: PAH — BSA conju-
gates (2 mg) were administered intramuscularly every
week, the first injection — in 0.5 ml of complete Freund’s
adjuvant (Sigma, USA), the second injection — in 0.5 ml
of incomplete Freund’s adjuvant, the third injection — in
1 ml of distilled water. Then, every 2 weeks booster injec-
tions of 1 mg of conjugate in 1 ml of distilled water were
administered. Blood was taken in two-month period after
the beginning of immunization once every two weeks.
Purification of hyperimmune antisera on affinity
columns. The PAH-HK ligands were immobilized on cya-
nogen bromide activated Sepharose 4B according to
the protocol of Pharmacia. After the antiserum sample
application, the column was washed with PBS, pH 7.5,
then Ab were eluted with 0.1 М glycine buffer, pH 2.5,
and extensively dialyzed against PBS. Concentration of
Ab in sample was determined with UV spectroscopy.
Enzyme immunoassay procedure. Affinity-puri-
fied Ab were pre-incubated with solutions of each five
conjugates PAHs-HK at 37 0C for 1 h. Five samples of
each anti-PAH Ab to each PAH were prepared. The
final concentration of Ab in the mixes was 4 μg/ml, the
concentration of PAH-HK conjugates — 100 μg/ml.
Preliminary experiments revealed that at these optimal
concentrations the binding of Ab (e. g., anti-Ac Ab)
with the corresponding coating conjugate PAH-HK
(Ac-HK on the plate) decreases by the same conjugate
(Ac-HK) more than by 75%. Then, these mixes were
added to wells of polystyrene microtiter plates with
coating antigens so that each anti-PAH Ab had the cor-
responding coating PAH-HK conjugate (e. g., mixes of
anti-Ac Ab with PAHs-HK were added to wells coated
ImmunologIcal Images of PolycyclIc aromatIc
Hydrocarbons
A.N. Glushkov1, M.V. Kostyanko2, T.P. Anosova1, E.G. Polenok1, S.A. Mun1, M.P. Anosov1, S.V. Cherno1
1Institute of Human Ecology, Siberian Branch of Russian Academy of Sciences, Kemerovo, Russia
2Kemerovo State University, Kemerovo, Russia
Aim: To develop experimental model for definition of immunological images of chemical carcinogens. Materials and Methods: The conjugates
of benzo[a]pyrene, benz[a]anthracene, anthracene, chrysene and pyrene with bovine serum albumin and yeast hexokinase were synthesized.
Rabbits were immunized by bovine serum albumin-hapten conjugates. Antibodies to each hapten were isolated from the serum by affinity
chromatography with the hapten-yeast hexokinase-Sepharose sorbents. The binding of each hapten with each antibody was determined by
competitive immunoassay. Results: The immunological images of all the investigated chemical compounds were described. Conclusion: The
model is proposed to determine the internal immunological images of anti-idiotypic monoclonal antibodies to chemical carcinogens.
Key Words: immunological image, chemical carcinogen, antibodies.
Received: February 14, 2006.
Correspondence: Tel/Fax: (3842) 545952
E-mail: ihe@kemtel.ru
Abbrereviations used: Ab – antibodies; Ac – anthracene;
Ba – benzaanthracene; Bp – benzoapyrene; BSA – bovine serum
albumin; Cr – chrysene; HK – yeast hexokinase; P – pyrene;
PAH — polycyclic aromatic hydrocarbons.
Exp Oncol 2006
28, 2, 166–168
Experimental Oncology 28, 166–168, 2006 (June) 16728, 166–168, 2006 (June) 167June) 167) 167 167
with Ac-HK). Ab-hapten binding was detected by the
standard technique using peroxidase-labelled goat
anti-rabbit IgG [5]. Absorbance values in wells with
Ab without adding the competitors were used as the
control. Then, parameters of Ab binding decrease were
calculated for each competitor and expressed in %.
Purifed polyclonal anti-PAH Ab were isolated from
rabbit antisera after the immunization of their PAH-BSA
conjugates on PAH-HK-Sepharose 4B affinity columns.
As a result, 0.14–1.2 mg of Ab was eluted from 1 ml of
antiserum. By means of the non-competitive ELISA it
was found out that the resulting Ab did not react with
BSA and HK, but they reacted with PAH-HK conjugates,
i. e. this method allowed us to get rid of accompanying
Ab to protein-carrier and to isolate Ab to hapten. The
parameters of binding decrease for each of Ab with the
corresponding coating PAH after pre-incubation of Ab
with the Ac, Ba, Bp, Cr and P competitors are submitted
in the Table. As one may see, the maximal binding de-
crease (more than 75%) is observed after the incubation
of each Ab with the PAH conjugate which was used for
immunization and affine purification of Ab. Competitive-
ness of other PAHs was always less pronounced.
Table. Parameters of binding for each Ab with corresponding coating PAH
after pre-incubation of Ab with the Ac, Ba, Bp, Cr and P competitors
Competitor Affinity-purified anti-PAH Ab / coating antigen PAH-HK
anti-Ac/Ac anti-Ba/Ba anti-Bp/Bp anti-Cr/Cr anti-P/P
Ac 86 19 35 14 7
Ba 23 77 71 45 12
Bp 16 55 87 54 17
Cr 15 50 78 93 20
P 19 50 72 33 76
The horizontal lines of the Table show the activity
of each of the five PAHs in separate pairs of anti-PAH
Ab and its inducing antigen, e.g., such a competitor
as Ac decreases the binding in the anti-Ac Ab/Ac-HK
pair by 86%. Further follow the parameters of binding
decrease for the following pairs: anti-Ba Ab/Ba-HK
(19%), anti-Bp Ab/Bp-HK (35%), anti-Cr Ab/Cr-HK
(14%) and anti-P Ab/P-HK (7%). Thus, the affinity of
Ac with all the anti-PAH Ab decreases from greater to
smaller in the following order:
Ac: Ac > Bp > Ba > Cr > P
The sequences for other PAH are as follows:
Ba: Ba > Bp > Cr > Ac > P
Cr: Cr > Bp > Ba > P > Ac
P: P > Bp > Ba > Cr > Ac
Bp: Bp > Ba > Cr > P > Ac
The affinity of each investigated PAH in relation to
each obtained anti-PAH Ab has characteristic specific
features. Firstly, the highest affinity of each of the PAH
to anti-PAH Ab induced by a corresponding PAH is de-
tected, and secondly, the affinity of Ac, Ba, Cr and P to
anti-Bp Ab. Each of these compounds is to some extent
a structural element of Bp, that is why, the interaction of
the binding site of Ab with PAH hapten is not sterically
hindered. The affinity of Ac to anti-Ba Ab (with a weaker
binding compared to anti-Bp Ab) comes third, probably,
due to a smaller size of the binding site of anti-Ba Ab
than that of anti-Bp Ab, though Ас is a structural element
of both Ba and Bp molecules. In all other cases lower
affinity of PAH to Ab with different specificity is due to
some unobservable differences in structure and size
between antigen and binding site of Ab.
Five diagrams of internal immunological images of
investigated PAH are presented in the Fig. 1. Each diagram
contains five axes, corresponding to decrease (in %) of
binding between affinity-purified anti-PAH Ab and antigen
inducing them in the presence of the PAH competitor.
figure. Internal immunological images of PAH. The axes show
the decrease of Ab binding to absorbed corresponding PAH after
preincubation of Ab with PAH-competitor (in %)
The most significant immunological similarity was
observed for Bp and Ba. Their diagrams are almost
indistinguishable, though the Bp molecule contains 5
fused aromatic rings, the Ba molecule — 4. The other
two compounds of this chemical group, namely Cr and
P, like the Ba molecule, consist of 4 rings, but they great-
ly differ from Ba and from one another as to the internal
immunological image. The internal immunological im-
age of three-ring Ac has even more prominent features,
completely different from the other four images.
Thus, insignificant differences in the structure of
chemical compounds with low molecular weight result
in significant differences in their internal immunologi-
cal images. Even PAH with the same amount of, but
differently fused, aromatic rings (Ba, Cr, P) possess
different internal immunological images.
Affinity-purified polyclonal Ab with high affinity to
Ac, Ba, Bp, Cr and P were obtained. Ab were shown to
react not only with the PAH — immunogene and affini-
ty-sorbent, but with structurally similar molecules. The
affinity of each of the five PAH to different purified Ab
was found to have its characteristic specific features.
The highest affinity of each PAH was observed for Ab,
induced by each PAH. Also, lower affinity of Ac, Ba, Cr and
P was found for anti-Bp Ab. It can be easily explained by
the fact that all substances include one part of Bp structure
and can freely interact with specific binding site of anti-Bp
Ab. It is evident that during immunization by Bp, lympho-
cyte clones with surface-cell immunoglobulin receptors,
more complimented to Bp, are activated at first stage. The
resulting Ab possess highest affinity to Bp, and at the same
time they are able to bind to PAH with lower molecular
weight, which are structural part of the Bp molecule.
It can be assumed that during immunization by
6-ring PAH with molecular weight, exceeding mo-
168 Experimental Oncology 28, 166–168, 2006 (June)
lecular weight of Bp, Ab with hight affinity to this PAH,
appreciable affinity to Bp and other types of PAH with
less molecular weight are most likely to be formed. It
is evident that increase in the number of anti-PAH Ab
allows us to reveal specific immunochemical proper-
ties of any investigated PAH in more precise detail.
All parameters of affinity of each PAH to wide spectra
of anti-PAH Ab in our model system can be called as
immunological image of PAH. The graphic represen-
tation of PAH immunological images is one of most
convenient illustraton of their features.
The established model the PAH immunological
images can be applied to define the internal immuno-
logical images of anti-idiotypic Ab to PAH and other
chemical carcinogens. If we investigate anti-idiotypic
Ab to PAH in terms of our model and compare its ability
to bindihg decrease of affinity-purified anti-PAH Ab with
corresponding PAH, its internal immunological image
can be observed. Chagnaud et al. [1, 2] used serum
gamma-globulin fractions, containing anti-Bp Ab, to
prepare monoclonal anti-idiotypic Ab. Meanwhile, it is
evident that in addition to anti-Bp Ab, other anti-PAH Abs
were also present in these fractions. Experimentaly it is
stated that cross-reactivity between even affinity-puri-
fied Ab with high specificity to corresponding PAH and
other structurally similar compounds occurs. Therefore,
the internal immunological image of Ab obtained by
Chagnaud et al. remained unaccounted.
Moreover, the technique of preparing monoclonal Ab
does not exclude the fact that obtained Ab does not pos-
sess predetermined specificity, i. e. the highest affinity to
inducing hapten. Li K et al. [7] studied the mAb 10C10,
prepared earlier against Bp by Gomes et al. [6]. They
shown that binding of mAb with fluorene 9 fold and with
naphthalene — 95 fold stronger then with Bp. Therefore,
to prepare monoclonal anti-idiotypic Ab to carcinogen, it
is more expedient to use the affinity-purified Ab for im-
munization. Thus, the probability of induction of required
clone increases. For screening clones, it is necessary
to use wide spectra of affinity-purified Ab together with
corresponding structurally similar compounds.
It allows us to choose the clone, producing anti-idioty-
pic Аb, which internal immunological image corresponds
to the structure of initial carcinogen with highest degree.
This patterns, revealed by authors, can be useful for
preparation of anti-carcinogenic vaccines, based on
the anti-idiotypic Ab. Apparently, initiated carcinogen,
intended for this purpose, must be the substance with
molecular weight lower than that of Bp, for example any
fjord-region of PAH [8]. In that case the corresponding
anti-idiotypic Ab will provide immune protection against
carcinogens with different chemical structure.
aKnoWledgments
This work was funded by the financial support of
Administration of Kemerovo region.
references
1. Chagnaud JL, Faiderbe S, Geffard F. Curative effects on
rat sarcomas obtained after a treatment combining two mono-
clonal antibodies. Anticancer Drugs 1994; 5: 361–6.
2. Chagnaud JL, Faiderbe S, Geffard F. Effects of a mono-
clonal anti-idiotypic antibody, internal image of benzo(a)pyrene,
on rat sarcomas. C R Acad Sci III 1993; 316: 1266–9.
3. Glushkov AN. New approaches to immunoprevention
and immunotherapy of neoplasms. Russ J Immunol 2002;
7: 219–28.
4. Glushkov AN, Anosova TP, Kostyanko MV, Anosov MP,
Polenok EG, Cherno SV, Mun SA. Antisera to polycyclic aro-
matic hydrocarbons and their application on the detection of
chemical carcinogens in blood sera of oncological patients.
Exp Oncol 2004; 26: 145–148.
5. Glushkov AN, Kostyanko MV, Cherno SV, Vasilchen-
ko IL. Synthеsis of polycyclic aromatic hydrocarbon-protein
conjugates for preparation and immunoassay of antibodies.
Russ J Immunol 2002; 7: 41–6.
6. Gomes M, Santella RM. Immunologic methods for the
detection of benzo[a]pyrene metabolites in urine. Chem Res
Toxicol 1990; 3: 307–10.
7. Li K, Chen R, Zhao B, Liu M, Karu AE, Roberts VA,
Li QX. Monoclonal antibody based ELISA for part-per-billion
determination of polycyclic aromatic hydrocarbons: effects of
haptens and formats on sensitivity and specificity. Anal Chem
1999; 71: 302–9.
8. Likhachov AJ, Savochkina IV. Fjord-region polycyclic
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(In Russian).
ИммунологИческИе образы полИцИклИческИх
ароматИческИх углеводородов
Цель: разработка экспериментальной модели для определения иммунологических образов химических канцерогенов.
Материалы и методы: синтезированы конъюгаты бензо[a]пирена, бенз[a]антрацена, антрацена, пирена и хризена с бычьим
сывороточным альбумином и дрожжевой гексокиназой. Кроликов иммунизировали конъюгатами с бычьим сывороточным
альбумином в качестве белка-носителя. Антитела к каждому из гаптенов выделяли из сыворотки аффинной хроматографией
на сорбентах гаптен-дрожжевая гексокиназа-Sepharose. Способность антител связываться с соответствующими гаптенами
определяли с помощью конкурентного иммуноанализа. Результаты: в результате исследования охарактеризованы
иммунологические образы исследуемых химических канцерогенов. Выводы: предложенная модель дает возможность определять
внутренние иммунологические образы антиидиотипических моноклональных антител к химическим канцерогенам.
Ключевые слова: иммунологический образ, химический канцероген, антитела.
Copyright © Experimental Oncology, 2006
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