Изменение аффинности антител в ходе развития гуморального иммунного ответа
Изучали процесс аффинного созревания антител в ходе развития гуморального иммунного ответа у мышей BALB/c на введение миоглобина мышцы лошади. Для проведения этих исследований использован новый метод определения аффинности высоко- и низкоаффинных антител, присутствующих в смеси. Применение этого мет...
Gespeichert in:
| Datum: | 2008 |
|---|---|
| Hauptverfasser: | , |
| Format: | Artikel |
| Sprache: | Russian |
| Veröffentlicht: |
Інститут молекулярної біології і генетики НАН України
2008
|
| Schriftenreihe: | Біополімери і клітина |
| Online Zugang: | https://nasplib.isofts.kiev.ua/handle/123456789/157624 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | Изменение аффинности антител в ходе развития гуморального иммунного ответа / С.А. Бобровник, М.А. Демченко // Біополімери і клітина. — 2008. — Т. 24, № 1. — С. 14-20. — Бібліогр.: 20 назв. — рос., англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
nasplib_isofts_kiev_ua-123456789-157624 |
|---|---|
| record_format |
dspace |
| spelling |
nasplib_isofts_kiev_ua-123456789-1576242025-02-09T12:09:01Z Изменение аффинности антител в ходе развития гуморального иммунного ответа Зміна афінності антитіл у ході розвитку гуморальної імунної відповіді Изменение аффинности антител в ходе развития гуморального иммунного ответа Бобровник, S.A. Демченко, М.А. Изучали процесс аффинного созревания антител в ходе развития гуморального иммунного ответа у мышей BALB/c на введение миоглобина мышцы лошади. Для проведения этих исследований использован новый метод определения аффинности высоко- и низкоаффинных антител, присутствующих в смеси. Применение этого метода позволило получить основные характеристики гуморального иммунного ответа в ходе его развития на Т-зависимый белковый антиген. Установлено, что аффинность антител, их количество, а также доля высокоаффинных специфических антител постепенно возрастают в процессе развития иммунного ответа. Вивчали процес афінного визрівання антитіл при розвитку гуморальної імунної відповіді у мишей BALB/c на введення міоглобіну м’язів коня. Для проведення цих досліджень використано новий метод визначення афінності високо- і низькоафінних антитіл, наявних у суміші. Застосування цього методу дозволило одержати основні характеристики гуморальної імунної відповіді в ході її розвитку на Т-залежний білковий антиген. Встановлено, що афінність антитіл, їхня кількість, а також частка високоафінних специфічних антитіл поступово зростають при розвитку імунної відповіді. The process of affinity maturation in the course of humoral immune response of BALB/c mice against horse muscle myoglobin was studied. For this purpose we used the method of determination of the affinity constants for high- and low-affinity antibodies, present in the mixture. Application of this method allowed us to determine the main characteristics of humoral immune response change in the course of the immune response against T-dependent protein antigen. It was shown that the affinity of antibodies, their quantity, and the proportion of high-affinity specific antibodies gradually increased during immune response development. 2008 Article Изменение аффинности антител в ходе развития гуморального иммунного ответа / С.А. Бобровник, М.А. Демченко // Біополімери і клітина. — 2008. — Т. 24, № 1. — С. 14-20. — Бібліогр.: 20 назв. — рос., англ. 0233-7657 https://nasplib.isofts.kiev.ua/handle/123456789/157624 577.27:616.097 DOI: http://dx.doi.org/10.7124/bc.00078B ru Біополімери і клітина application/pdf application/pdf Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
Russian |
| description |
Изучали процесс аффинного созревания антител в ходе развития гуморального иммунного ответа у мышей BALB/c на введение миоглобина мышцы лошади. Для проведения этих исследований использован новый метод определения аффинности высоко- и низкоаффинных антител, присутствующих в смеси. Применение этого метода позволило получить основные характеристики гуморального иммунного ответа в ходе его развития на Т-зависимый белковый антиген. Установлено, что аффинность антител, их количество, а также доля высокоаффинных специфических антител постепенно возрастают в процессе развития иммунного ответа. |
| format |
Article |
| author |
Бобровник, S.A. Демченко, М.А. |
| spellingShingle |
Бобровник, S.A. Демченко, М.А. Изменение аффинности антител в ходе развития гуморального иммунного ответа Біополімери і клітина |
| author_facet |
Бобровник, S.A. Демченко, М.А. |
| author_sort |
Бобровник, S.A. |
| title |
Изменение аффинности антител в ходе развития гуморального иммунного ответа |
| title_short |
Изменение аффинности антител в ходе развития гуморального иммунного ответа |
| title_full |
Изменение аффинности антител в ходе развития гуморального иммунного ответа |
| title_fullStr |
Изменение аффинности антител в ходе развития гуморального иммунного ответа |
| title_full_unstemmed |
Изменение аффинности антител в ходе развития гуморального иммунного ответа |
| title_sort |
изменение аффинности антител в ходе развития гуморального иммунного ответа |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2008 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/157624 |
| citation_txt |
Изменение аффинности антител в ходе развития гуморального иммунного ответа / С.А. Бобровник, М.А. Демченко // Біополімери і клітина. — 2008. — Т. 24, № 1. — С. 14-20. — Бібліогр.: 20 назв. — рос., англ. |
| series |
Біополімери і клітина |
| work_keys_str_mv |
AT bobrovniksa izmenenieaffinnostiantitelvhoderazvitiâgumoralʹnogoimmunnogootveta AT demčenkoma izmenenieaffinnostiantitelvhoderazvitiâgumoralʹnogoimmunnogootveta AT bobrovniksa zmínaafínnostíantitíluhodírozvitkugumoralʹnoíímunnoívídpovídí AT demčenkoma zmínaafínnostíantitíluhodírozvitkugumoralʹnoíímunnoívídpovídí |
| first_indexed |
2025-11-25T22:56:15Z |
| last_indexed |
2025-11-25T22:56:15Z |
| _version_ |
1849804854184116224 |
| fulltext |
STRUCTURE AND FUNCTIONS OF BIOPOLYMERS
Affinity maturation of antibodies in the course of
humoral immune response
S.A. Bobrovnik, M.A. Demchenko
Palladin Institute of Biochemistry NAS of Ukraine
9 Leontovicha Street, Kyiv, 01601, Ukraine
sab@biochem.kiev.ua
The process of affinity maturation in the course of humoral immune response of BALB/c mice against horse
muscle myoglobin was studied. For this purpose we used the method of determination of the affinity
constants for high- and low-affinity antibodies, present in the mixture. Application of this method allowed us
to determine the main characteristics of humoral immune response change in the course of the immune
response against T-dependent protein antigen. It was shown that the affinity of antibodies, their quantity,
and the proportion of high-affinity specific antibodies gradually increased during immune response
development.
Keywords: affinity, affinity maturation, non-linear regression
Introduction. The development of immune response
to various protein antigens is known to result in the
change in the affinity of antibodies, which is higher at
later stages of immune response compared to its initial
stages [1-5]. Reverse effect may take place only in case
of some disorders in the development of immune
response, in the course of some diseases or when the
organism is immunized with extremely high dosages of
antigen, which leads to exhaustion of corresponding
clones of high-affinity B-lymphocytes. In other cases
the affinity of antibodies increases, especially after
repeated immunizations.
It is noteworthy that determination of affinity of
antibodies in blood serum, containing a great number of
clones of similar specificity but different affinity, is a
difficult task. In most cases the use of current
investigation methods allows defining only the average
value, which does not show the exact amount and type
of antibodies (high- or low-affinity), present in the
investigated mixture. For instance, the method,
suggested in [6], and its modification, described by
Stevens in [7], allow accurate determination of the
affinity of only one clone of bivalent antibodies, using
ELISA. In case if the investigated mixture contains two
or more kinds of antibodies, including high- and
low-affinity ones, the result of determining affinity
depends significantly on the ratio of each kind of these
antibodies as well as on the range of concentrations of
competing antigen, used in the experiment [8-11].
The aim of current work was to determine affinity
of two kinds of antibodies, present in the mixture, and
to find the correlation between the amounts of high- and
low-affinity antibodies in investigated samples. It
allowed us to set the task of quantitative estimation of
parameters, reflecting the process of development of
immune response to protein antigen, which results in
maturation of affinity of antibodies and change in the
14
ISSN 0233-7657. Biopolymers and cell. 2008. Vol. 24. N 1.
Ó S.A. BOBROVNIK, M.A. DEMCHENKO, 2008
ratio of concentrations of high- and low-affinity
antibodies.
Theory. To determine the affinity of one kind of
antibodies using ELISA, Friguet et al. [6] elaborated
the method which gained wide popularity due to the
simplicity of its application and accuracy of obtained
data. The authors showed that in case of excess in
competing antigen in the antigen-antibody mixture,
there is a linear dependency between the ratio of total
amount of antibodies to the amount of antibodies,
blocked by antigen, and the reverse concentration of
antigen
A
A A
K
l
i
d
i
0
0
1
-
= + , (1)
where Kd – dissociation constant, i.e. value, inverse to
the value of affinity; A0 – staining of cells in the
absence of antigen; Ai – staining of cells in the presence
of competing antigen, whose concentration equals li.
The diagram of this dependency is a straight line
with the slope, which is equal to the value of affinity
constant. Therefore, this diagram allows determining
affinity of interaction of antibodies and antigen.
Later Stevens [7] voiced an opinion that equation
(1), suggested by Friguet et al., can be related only to
univalent antibodies, while to consider bivalent
antibodies, one should define the square root of the left
side of equation (1), so the proper equation will be
A
A A
K
l
i
d
i
0
0
1
-
= + . (2)
We have obtained equation (2) strictly
mathematically which proved [8, 9] the correctness of
Stevens’ remark [7]. Theoretical calculations (Fig.1, a)
and experimental data evidence to the correctness of
equation (2) as well.
At the same time, in case if the investigated sample
of antibodies contains two kinds of antibodies of
similar specificity, and their affinity differs
considerable, the diagram will present non-linear
dependency in the form of a convex curve instead of
linear dependency between the values of A
A A i
0
0 -
and l/li, presented by a straight line (Fig.1, b). To
determine affinity of both kinds of antibodies and the
ratio between their amounts in the investigated sample,
we suggested new coordinates, where presentation of
data on the amount of free antibodies in the mixture
with different concentrations of competing antigen is
shown in the form of concave curve (Fig.2, a). The
solution of these equations , and thus, the determination
of quantitative values of parameters under question, i.e.
values of K1 and K2, A1 and A2 [8-10] becomes possible
after finding the slope angles of asymptotes to this
curve, as well as cross-points of asymptotes with axes
of coordinates, and introducing the found values into
suggested four equations.
The drawback of the mentioned method is the use of
a wide range of concentrations of the competing
antigen with the purpose of increasing the accuracy of
determination of required characteristics of the mixture
of antibodies. Only sufficient range of concentrations
of antigen allows drawing experimental curves of
binding which are rather close to axes of coordinates
and thus allow obtaining sufficiently accurate required
values of the affinity of both kinds of antibodies and the
ratio of their concentrations. Unfortunately, these
experiments are technically complicated, and the
accuracy of determination decreases significantly when
one defines staining of cells with very low intensity.
Therefore, we have suggested a new method [11,
12], which is rather simple, yet allows obtaining more
accurate data concerning the affinity of two kinds of
antibodies and the ratio of their amounts in samples. It
is based on the method of non-linear regression for
calculation of parameters of the curve of binding using
the known equation due to modern methods of
computational mathematics, e.g. Origin 75 software.
We have shown previously that the dependency of
intensity of staining of wells Ai, defined with ELISA (in
case if staining of wells is proportional to the
concentration of free antibodies in the antigen-antibody
mixture) on the concentration of competing antigen li
can be presented by the following equation (Fig.2, b):
A
A l K
l K
A l K
l K
i
i
i
i
i
=
+
+
+
+
+
1 1
1
2
2 2
2
2
1 2
1
1 2
1
( )
( )
( )
( )
, (3)
where K1 and K2 – affinity constants of both kinds of
antibodies; A1 and A2 – staining of wells, which would
be obtained in case of using each kind of antibodies
separately in the absence of competing antigen.
15
AFFINITY OF ANTIBODIES IN THE COURSE OF HUMORAL IMMUNE RESPONSE
Having found values of A1 at different known
values of competing antigen li via experiment, it is
possible to apply the method of non-linear regression to
find the values of K1 and K2, as well as A1 and A2 which
would correspond to experimental curve for its
coincidence to the curve, presented by equation (3) in
the best possible way. Since values of A1 and A2 are
proportional to the concentration of each of antibodies
in the mixture, it is possible to not only define the
affinity of both antibodies K1 and K2 but also to find the
ratio of concentrations of these antibodies, which is
equal to the ratio of A1 to A2, or a part of each kind of
antibodies, which equals A1/(A1 + A2) and A2 (A1 + A2).
Materials and Methods. Horse muscle myoglobin
was used as antigen (Sigma, USA). Six mice of
BALB/c line (female mice, weight about 22-25 gram)
were immunized intravenously with 250 µg/per mouse
of myoglobin, diluted in the physiological solution.
Immunization was performed with the similar dose of
mentioned antigen three times, with the interval of three
weeks. In 10 days after each immunization
approximately 250 µl of blood were taken from the tail
veins of mice to obtain serum. Sera of three mice were
combined in two pools, consisting of three separate
sera. Antisera to myoglobin, obtained by the
aforementioned method, were frozen.
Myoglobin was sorbed on 96-cell Mai Sorp plates
(Nunc, Denmark), via incubating 110 µl/cell of
myoglobin solution (30 µg/ml in 0.1% solution of
ammonium bicarbonate, pH 8.5) in plates at 4°C for 18
hours. Prior to using plates were thoroughly washed of
non-bound myoglobin, and introduced with 100 µl/cell
of investigated mixture, consisting of diluted antiserum
and some amount of myoglobin. Plates were incubated
with investigated samples of antisera and antigen for
45-50 min at constant shaking, then the solutions were
removed from the cells and plates were washed with
running water. ELISA was applied to determine the
amount of antibodies of IgG class, bound to myoglobin,
sorbed on the plate, i.e. we determined the amount of
antibodies, one or two paratopes of which were not
occupied by the soluble antigen.
To determine the dilution of antisera, optimal for
the determination of affinity of antibodies by
16
BOBROVNIK S.A., DEMCHENKO M.A.
Fig.1 Theoretical curves of antigen binding
by bivalent antibodies, presented in
coordinates Y = =
A
A Ai
0
0 -
against l/li: a –
linear dependency between values of
A
A Ai
0
0 -
and l/li for one kind of antibodies
with the affinity constant K = 1.0•108 M-1; b
– non-linear dependency between the
values of
A
A Ai
0
0
-
and l/li for two kinds of
antibodies in equal concentrations with the
affinity constants K1 = 1,0×108 Ì–1 è K2 =
= 1,0×106 Ì–1
mentioned methods, sera were titrated in 0.2% solution
of BSA in buffered physiological solution, containing
0.1% twin-20 (TBF). Dilutions of antiserum, providing
directly proportional decrease in the intensity of cell
staining at the decrease in antiserum concentration,
were further used in the experiments of determining the
affinity of antibodies [6].
Aliquots of 0.2 ml of (1:50) – (1:150) times diluted
antisera (according to the data of the preliminary
experiment of titration) were 1:1 mixed with the
solutions of myoglobin of different concentration (from
2.5•10-7 to 6.4•10-5 M) in 0.2% solution of BSA in TBF.
Samples were incubated at room temperature for 18
hours, then 0.1 ml of each sample of mixture (in three
replications) were introduced into the wells of
immunological plates with sorbed myoglobin, plates
were incubated with these samples for 45-50 min at
constant shaking, then samples were removed from the
wells, plates were washed and the relative amount of
bound antibodies was determined using ELISA. The
values of staining intensity for each sample were
defined via calculation of the average value from three
measurements.
Method of non-linear regression was used to
determine the values of K1, K2, A1, and A2, then the ratios
A1/(A1 +A2) and A2/(A1 +A2) were calculated i.e. the ratio
of concentrations of high- and low-affinity antibodies
in the investigated mixture was found.
Results and Discussion. Fig.3, a presents the
results of titration of antisera to myoglobin with
ELISA, obtained after one-, two-, and three-time
intravenous immunization of mice with myoglobin.
Dilution in more than 50 times leads to the occurrence
of linear dependencies between the concentration of
antiserum and intensity of well staining. At the same
time concentration of IgG antibodies in antiserum after
two-times immunization is approximately 1.5 times
higher compared to that after one-time immunization,
and the concentration of antibodies after three-times
immunization is approximately 2 times higher
compared to that after two-times immunization.
Therefore, to determine affinity of IgG antibodies,
antisera were diluted 50, 75, and 150 times to get
approximately equal concentrations of antibodies in the
obtained samples.
Fig.3, b shows results evidencing to the dependence
of the amount of free IgG antibodies in the mixture with
antigen on the concentration of antigen. Since each of
the curves, presented in this Figure, is described with
equation (3), application of Origin 75 software allows
determining values of K1, K2, A1, and A2, the most
appropriate to the adjustment of obtained experimental
data to the theoretical curve, described by equation (3).
The required values are presented in Table.
The data of Table demonstrate that experiments
proved gradual increase in the affinity of forming IgG
antibodies in the process of development of immune
response to T-dependent protein antigens. This pattern
is not surprising, since affinity maturation of immune
response is known to include first switching synthesis
17
AFFINITY OF ANTIBODIES IN THE COURSE OF HUMORAL IMMUNE RESPONSE
Fig.2 Non-linear dependency of the amount of free antibodies (i.e.
antibodies, one or two paratopes of which are not blocked by
antigen), present in the mixture with antigen in equilibrium, on the
concentration of antigen (a) and the same dependency in the
coordinates Y =
A A A A A
l
i i
i
0 0 0 710
- + -
× -
( )
against the value A0 – Ai
(b). Both theoretical curves are calculated for the following
parameters of antigen-antibody interaction: K1 = 1.0•108 M-1; K2 =
1.0•106 M-1; range of antigen concentrations li = 1.0•10-9 – 2.62•10-4
M, A1 = 1.0; A2 = 1.0; A0 = A1 + A2
of antibodies from IgM class to IgG [8], then gradual
selection of clones of B-lymphocytes, producing
antibodies with higher affinity [2-5, 13, 14].
This process, called maturation of affinity of
antibodies, consists of both somatic mutations,
responsible for the structure of recognition centre of
antibody, and subsequent selection of clones,
producing antibodies with higher affinity [5, 15–18].
Therefore this process is considered to resemble
evolution according to Darwin, since the first stage is
mutation of the corresponding gene, and the second one
is selection of clones with the most advantageous
mutations [19].
It should be noted that application of suggested
method allows not only determining the tendency
towards increase in similarity between antibodies and
antigen in the process of immune response, but also to
track the dynamics of appearing of antibodies with
higher affinity as well as ratio of their amount to
low-affinity antibodies. Table demonstrates that in 10
days after one-time immunization of horse muscle
myoglobin sera of mice has only about 10% of
relatively high-affinity antibodies (K1 = 1.62•106 M-1)
and about 90% – of low-affinity antibodies (K2 =
1.17•104 M-1).
After repeated immunization of mice with
myoglobin the amount of high-affinity antibodies is
one third of all antibodies, and the affinity of these
high-affinity antibodies increases to 8.74•106 M-1, i.e.
exceeds that of high-affinity antibodies after one-time
immunization about 5 times. The affinity of
low-affinity antibodies (K1 = 1.62•106 M-1) is also
higher compared to the first immunization though in
this case the difference is only about 1.5 times.
Finally, after the third immunization the ratio of
high-affinity antibodies increases even more and is
18
BOBROVNIK S.A., DEMCHENKO M.A.
Fig.3 Experimental curves of dependency of free IgG
antibodies to myoglobin on the dilution of serum or on
concentration of myoglobin in the mixture with
antibodies: a – influence of dilution of antisera to
myoglobin on the amount of antibodies, bound to
myoglobin, which was sorbed on the immunological
plate (1 – one-time; 2 – two-time; 3 – three-time); b –
effect of myoglobin concentration in the mixture with
antisera to myoglobin on the amount of antibodies in
the myoglobin-antiserum mixture, where one or two
paratopes are not bound to myoglobin and are free (1 –
one-time; 2 – two-time; 3 – three-time)
close to 50%. Their affinity (K1 = 1.23•107 M-1) is also
higher compared to the affinity of antibodies after
two-times immunization. Low-affinity antibodies after
three-time immunization (K2 = 4.49•104 M-1) have
approximately three times higher affinity to myoglobin
compared to similar antibodies after two-times
immunization. Therefore, the process of development
of immune response is characterized not only by the
increase in the ratio of high-affinity antibodies
compared to low-affinity antibodies, but also by the
increase in absolute values of affinity of pools of both
high-affinity and low-affinity antibodies.
It is evident that results obtained are in complete
agreement with numerous data [1–5, 13–20]
concerning gradual qualitative change in characteristics
of produced antibodies in response to repeated
immunization of the organism with foreign protein
antigen. Besides, the use of elaborated method of
estimating affinity of high- and low-affinity antibodies,
present in the mixture, allowed obtaining quantitative
characteristics of this process. The data of Fig.3, a,
proving the increase in total amount of
myoglobin-specific antibodies, which appear in blood
flow in the process of immune response to intravenous
immunizations with myoglobin, as well as obtained
additional data on the characteristic of produced
antibodies allow explaining the events in the course of
development of humoral immune response to protein
antigens.
The results of our investigation evidence to the
importance of determining both the affinity of not less
than two kinds of antibodies of the same specificity in
case of their presence in the mixture, and the ratio
between concentrations of these antibodies. Current
work testifies to the significance of elaborated method
of estimating the affinity of high- and low-affinity
antibodies, present in the mixture in unknown
proportions.
Ñ. À. Áîá ðîâ íèê, Ì. À. Äåì ÷åí êî
Èçìå íå íèå àô ôèí íîñ òè àí òè òåë â õîäå ðàç âè òèÿ ãó ìî ðàëü íî ãî
èì ìóí íî ãî îò âå òà
Ðå çþ ìå
Èçó ÷à ëè ïðî öåññ àô ôèí íî ãî ñî çðå âà íèÿ àí òè òåë â õîäå ðàç âè -
òèÿ ãó ìî ðàëü íî ãî èì ìóí íî ãî îò âå òà ó ìû øåé BALB/c íà ââå -
äå íèå ìè îã ëî áè íà ìûø öû ëî øà äè. Äëÿ ïðî âå äå íèÿ ýòèõ
èñ ñëå äî âà íèé èñ ïîëü çî âàí íî âûé ìå òîä îïðå äå ëå íèÿ àô ôèí -
íîñ òè âû ñî êî- è íèç êî àô ôèí íûõ àí òè òåë, ïðè ñó òñòâó þ ùèõ â
ñìå ñè. Ïðè ìå íå íèå ýòî ãî ìå òî äà ïî çâî ëè ëî ïî ëó ÷èòü îñíîâ -
íûå õà ðàê òå ðèñ òè êè ãó ìî ðàëü íî ãî èì ìóí íî ãî îò âå òà â õîäå
åãî ðàç âè òèÿ íà Ò-çà âè ñè ìûé áåë êî âûé àí òè ãåí. Óñòà íîâ ëå íî,
÷òî àô ôèí íîñòü àí òè òåë, èõ êî ëè ÷åñ òâî, à òàê æå äîëÿ âû ñî -
êî àô ôèí íûõ ñïå öè ôè ÷åñ êèõ àí òè òåë ïî ñòå ïåí íî âîç ðàñ òà þò
â ïðî öåñ ñå ðàç âè òèÿ èì ìóí íî ãî îò âå òà.
Êëþ ÷å âûå ñëî âà: àô ôè íè òåò, àô ôèí íîå ñî çðå âà íèå, íå ëè -
íåé íàÿ ðåã ðåñ ñèÿ.
REFERENCES
1. Ðîéò À. Îñíî âû èì ìó íî ëî ãèè.– Ì.: Ìèð, 1991.– 327 ñ.
2. Azur Z., Mazor G., Meilijson I. Maturation of the humoral
immune response as an optimization problem // Proc. Biol.
Sci.–1991.–245.–P. 147–150.
3. MacLennan I. C., Liu Y. J., Johnson G. D. Maturation and
dispersal of B-cell clones during T cell-dependent antibody
responses // Immunol. Rev.–1992.–126.–P. 143–461.
4. Forsdyke D. R. The origins of the clonal selection theory of
immunity as a case study for evaluation in science // FASEB
J.–1995.–9.–P. 164–166.
5. Kelsoe G. Life and death in germinal centers (redu) //
Immunity.–1996.–4.–P. 107–111.
6. Friguet B., Chaffotte A. F., Djavadi-Ohaniance L., Goldberg
M. E. Measurements of the true affinity constant in solution
of antigen-antibody compleões by enzyme-linked
19
AFFINITY OF ANTIBODIES IN THE COURSE OF HUMORAL IMMUNE RESPONSE
Values of affinity constants of high- and low-affinity antibodies to myoglobin in sera of BALB/c mice in 10 days after intravenous
immunization of mice with horse muscle myoglobin
Immunization K1 K2 A1/(A1 + A2), % A2/(A1+ A2), %
One-time
1,62×106
(±3,22×105)
1,17×104
(±9,49×102)
9,9 90,1
Two-time
8,74××106
(±2,17×106)
1,62×104
(±4,71×103)
33,3 66,7
Three-time
1,23×107
(±1,37×106)
4,49×104
(±4,68×103)
46,5 53,5
immunosorbent assay // J. Immunol. Meth.–1985.–77.–
P. 305–319.
7. Stevens F. J. A modification of an ELISA-based procedure
for affinity determination: correction necessary for use with
bivalent antibody // Mol. Immunol.–1987.–24.–P. 1055–
1060.
8. Áîá ðîâ íèê Ñ. À. Îïðå äå ëå íèå àô ôèí íîñ òè àí òè òåë ñ ïî -
ìîùüþ ELISA // Óêð. áè î õèì. æóðí.–1999.–71, ¹ 6.–
Ñ. 90–102.
9. Bobrovnik S. A. ELISA-based method for determining the
affinity of bivalent antibodies of two specificities in a miture
// Ukr. biokhem. zh.–2000.–72, N 3.–P. 133–141.
10. Bobrovnik S. A. Determination of antibody affinity by
ELISA. Theory // J. Biochem. and Biophys. Meth.–2003.–
57.–Ð. 213–236.
11. Áîá ðîâ íèê Ñ. À. Îïðå äå ëå íèå àô ôèí íîñ òè âû ñî êî- è íèç -
êî-àô ôèí íûõ àí òè òåë, íà õî äÿ ùèõ ñÿ â ñìå ñè, ïðè ïî ìî -
ùè ELISA è ìå òî äà íå ëè íåé íîé ðåã ðåñ ñèè // Óêð. áè î õèì.
æóðí.–2005.–77, ¹ 3.–Ñ. 155–161.
12. Bobrovnik S. A., Stevens F. J. Deconvolution of antibody
affinities and concentrations by non-linear regression
analysis of competitive ELISA data // J. Immunol. Meth.–
2007.–328.–Ð. 53–58.
13.Cauerhff A., Goldbaum F. A., Braden B. C. Structural
mechanism for affinity maturation of an anti-lysozyme
antibody // Proc. Nat. Acad. Sci. USA.–2004.–101.–Ð. 3539–
3544.
14.Adams C. L., MacLeod M. K. L., Milner-White E. J., Aitken R.,
Garside P., Scott D. I. Complete analysis of the B-cell
response to a protein antigen, from in vivo germinal centre
formation to 3-D modelling of affinity maturation //
Immunology.–2003.–108.–P. 274–287.
15.Celada F., Seiden P. E. Affinity maturation and
hypermutation in a simulation of the humoral immune
response // Eur. J. Immunol.–1996.–26.–P.1350–1358.
16. Maclennan I. C. M. Germinal centre // Annu. Rev. Immunol.–
1994.–12.–P. 117–139.
17. Berek C., Berger A., Apel M. Maturation of the immune
response in germinal centres // Cell.–1991.–67.–P. 1121–
1129.
18. Neuberger M. S., Milstein C. Somatic hypermutation // Curr.
Opin. Immunol.–1995.–7.–P. 248–254.
19. Kelsoe G. V(D)J hypermutation and receptor revision:
coloring outside the lines // Curr. Opin. Immunol.–1999.–
11.–P. 70–75.
20. Furukawa K., Akasako-Furukawa A., Shirai H., Nakamura
H., Azuma T. Junctional amino acids determine the
maturation pathway of an antibody // Immunity.–1999.–11.–
P. 329–338.
UDC 577.27:616.097
Received 27.04.07
20
BOBROVNIK S.A., DEMCHENKO M.A.
|