Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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P 34 20 926.3
METHOD AND REAGENT FOR THE DIFFERENTIATED DETERMINATION
OF ISOENZYMES OF ALKALINE PHOSPHATASE
AND MONOCLONAL ANTIBODY S~ITABLE FOR THIS PURPOSE
Alkaline phosphatase (orthophosphoric monoester
phosphohydrolase) catalyzes the hydrolysis of phosphoric acid
monoesters at alkaline pH levels. This enzyme occurs in many
tissues. Tissue-specific forms (isoenzymes) exist in the
liver, in the bones, in the small intestine~ in the kidneys
and in the placenta, for example. The isoenzymes are
released from these organs to the blood plasma. The
characteristic properties of the particular isoenzymes are
preserved in the transition from the producing organ to the
blood plasma.
The blood plasma of healthy persons contains mainly the liver
isoenzyme and the bone isoenæyme. The determination of the
total activity of the alkaline phosphatase as well as
especially the activity of the liver and bone isoenzymes is
of especial diagnostic importance in the study of diseases of
the liver and of the bone system. The diseases produce an
increase in alkaline phosphatase activity in the plasma,
since under these conditions alkaline phosphatase is released
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to an increased extent by the liver or bones, as the case may
be. The increase in the alkaline phosphatase activity of the
liver or bone isoenzyme in the plasma can accordingly serve
for the recognition of a disease of these tissues.
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The differentiation of the liver isoenzyme and of the bone
isoenzyme from other alkaline phosphatase isoenzymes~ e.g.,
those from the small intestine, from placenta, from kidneys
and from gall bladder, is easily possible since these tissue-
specific forms are clearly dlstinct in their chemical,
physical and immunological properties. Methods are known for
the separation and determination of these isoenzymes and are
based, for example, on the different behavior of the
isoenzymes against inhibitors, on differences in electrical
charge etc. For example, differences in electrophoretic
mobility in alkaline buffer systems are used $or the
separation of these isoenzymes.
Lesser differences, however, are to be observed between the
llver isoenzyme and the bone isoenzyme. In particular, the
liver and bone isoenzymes o$ alkaline phosphatase differ but
slightly with regard to heat stability and in response to
inhibitors (for example to urea). These differences do not
su$fice for the adequate and simple quantification o$ one
isoenzyme form in a mixture of both isoenzymes. Slightly
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more pronounced are the differences in the elec-trical
charge. These differences are utilized for the
~eparation of bone isoenzyme and of liver isoenzyme
and thus for the selective determination of the two
isoenzymes. These methods of separa-tion as well as
the methods of determination based -thereon require a
great amount of time and labor, and also call for a
great investment in apparatus.
There still is a need for a possibility of separating
and dif~erentiating the two isoenzymes quickly and
reliably in a simple manner -to the extent that at
leas-t one of -the two isoenzymes can be determined
selectively. The activity of the other isoenzyme
could then easily be determined as the difference
between the total activity of the mix-ture, which can
be determined by ]cnown methods, and the measured bone
or l.iver isoenzyme activity.
The lnvention seeks -to devise a new method and
reagent whereby a differentia-tion of the liver
isoenzyme and oE the bone isoenzyme of alkaline
phosphatase would be made possib:Le, as well as a
selective determination of the differentiated iso-
enzyme in a body Eluid. This may be achieved by the
method of the invention, in which the differentiation
is performed by means of a monoclonal antibody which
is directed against one of the two isoenzymes
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and shows only a slight cross-reaction with the other enzyme.
The method of the invention for the differentiation of the
bone isoenzyme and of the liver isoenzyme of al~aline
phosphatase can be performed on the one hand with a
monoclonal antibody which predominantly binds the liver
isoenzyme and reacts but slightly with the bone isoenzyme.
It is in like manner also possible to use a monoclonal
antibody which predominantly binds the bone isoenzyme and
shows only a slight cross reaction with the liver isoenzyme.
It has been possible to show that it is surprisingly possible
to find a monoclonal antibody which predominantly binds one
of the two isoenzymes and has only a slight cross-reactivity
to the other isoenzyme. This was not to be expected, since
precisely the liver isoenzyme and the bone isoenzyme are so
largely similar in their property that an imrnunological
differentiation of these two isoenzymes has not been possible
heretofore.
An additional subject matter of the invention is therefore a
new monoclonal antibody which is directed either against the
liver isoenzyme or against the bone isoenzyme and has only
slight cross-reactivity to the other isoenzyme. The
monoclonal antibody in accordance with the invention can
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accordingly be directed against the liver isoenzyme and have
only a slight cross-reactivity to the bone isoenzyme. It can
also, however, predominantly bind the bone isoenzyme and
cross-react but slightly with the liver isoenzyme. The cross-
reactivity with the other isoenzyme in either case can be 20%
or less.
The monoclonal antibody of the invention can be obtained in a
conventional manner. For this purpose experimental animals
are immunized with highly purified alkaline phosphatase ~rom
liver or bone. B lymphocytes of the immunized animals thus
obtained are fused with transforming agents. The primary
cultures of hybrid cells thus formed are cloned. In each
case, those cultures are further processed which, in a
suitable test process, e.g., an enzyme immunoassay (ELISA
process), react positively against the isoenzyme against
which the tested monoclonal antibody is to be directed, and
negatively with the other isoenzyme. A hybridoma cell line
is thus obtained which produces the monoclonal antibody in
accordance with the invention. By known methods this cell
line can be cultivated and the monoclonal antibody produced
by it can be isolated.
The animals especially suitable ~or the immunization are
particularly rats and mice. The immunization is performed
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with highly purified alkaline phosphatase from liver or
bones. For this purpose the enzyme, preferably in
combination with an adjuvant, is administered in a
conventional manner to the host animal. Aluminum hydroxide
toget~er with Bordatella pertussis or Freund's adjuvant are
used preferentially as adjuvant.
The immunization is performed preferably over several months
with at least 4 immunizations (intraperitoneal injection).
After the immunization is performed the B-lymphocytes of the
immunized animals are fused by conventional methods with
transforming agents. Examples of transforming agents which
can be used in the scope of the invention are myeloma cells,
transforming viruses such as Epstein-Barr virus for example,
or the agents described in German Offenle~unqsschrift 32 45
665. The fusing is performed by the known method of Koehler
and Milstein (Nature 2S6 [1975] pp. 495~997). The primary
cultures of hybrid cells thus formed, if they contain
antibody of the desired specificity, are cloned in a
conventional manner, using, for example, a commercial cell
sorter, or by "limiting dilution", and the clones obtained,
which form the desixed monoclonal antibody, are cultivated.
On the basis of the cancer-like growth of the hybrid cells,
they can be further cultivated indefinitely and produce the
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desired monoclonal antibody in any desired amount.
A hybridoma cell line obtained in this manner is cell line B
4 - 50, which has been filed in the American Type Culture
Collection under the number ATCC 8571. This cell line
produces a monoclonal antibody, a1so called B 4 - 50, which
is directed against the liver isoenzyme of alkaline
phosphatase and has a cross-reactivity to bone isoenzyme of
approximately 20%.
For the determination method of the invention, the monoclonal
antibodies can be used as they are or fragments having the
corresponding immunological properties (Fab fragments) can be
used. The term, "monoclonal antibodies", therefore is to be
understood to include both the whole antibodies and the
fragments.
The monoclonal antibodies thus obtainable are directed either
against the liver isoenzyme or against the bone isoenzyme of
alkaline phosphatase, and have only a slight cross-reaction
to the other isoenzyme. In a sample which contains both the
liver isoenzyme and the bone isoenæyme, they are therefore
outstandingly suitable for binding preferentially one of
these two isoenzymes. In this manner it is possible to
differentiate between the two isoenzymes. They can be
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determined selectively. This is accomplished preferably by
determining the phosphatase activity of the bound isoenzyme
content, without that of the dissolvad isoenzyme content.
With the aid of a calibration curve established with known
isoenzyme concentrations, the content of the isoenzyme
against which the monoclonal antibody used is directed can be
determined. The activity of the other enzyme is established
as the difference between the total activity of the alkaline
phosphatase of the sample and the measured isoenzyme
activity.
The determination of the alkaline phosphatase activity is
performed by the methods known for this purpose.
For the performance of the process of the invention, the
monoclonal antibody is fixed preferably on a solid support,
such as for example immunosorptive paper, on activated glass
particles or latex particles, on microtitration plates, or on
the surface of plastic tubes. In this manner the enzyme
against which the monoclonal antibody used is directed is
bound to the support, i.e., the solid phase, and can there be
20- determined as described above.
It is also possible, however, to separate the bound
enzyme from th~ liquid phase and to determine the remaining
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alkaline phosphatase activity in the liquid phase. With the
aid of a calibration curve established, again, with known
enzyme concentrations J it is possible thus to determine the
isoenzyme content against which the monoclonal antibody used
is not directed. From the difference between the total
alkaline phosphatase activity and the measured isoenzyme
content the content of the other iso~nzyme can be found.
In accordance with the invention, an antibody preparation can
be used which consists of several monoclonal antibodies which
have been produced by several different clones.
The complex formed from the monoclonal antibody and its
antigen, the liver isoenzyme or bone isoenzyme of the
alkaline phosphatase, is soluble. Its separation from the
liquid of the sample can also be performed by adding an
additional antibody against the monoclonal antibody (i.e., an
anti-antibody). An insoluble complex is thus formed from the
liver or bone isoenzyme of alkaline phosphatase, the
monoclonal antibody against this isoenzyme, and the anti-
antibody.
A desirable embodiment of the process of the invention
consists in first forming a complex from monoclonal antibody
and the anti-antibody, and then adding these to the sample
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liquid being tested. Alternatively, however, it is possible
first to add only the monoclonal antibody to the sample
liquid, and, after an incubation period sufficient for the
formation of the antigen-antibody complex with the liver or
bone isoenzyme of alkaline phosphatase, to add the anti-
antibody with the formation of the insoluble complex. For
the embodiment of the process o the invention using an anti-
antibody, all anti-antibodies directed against the monoclonal
antibody are hasically suitable. If the production of the
monoclonal antibody of the invention i5 performed in mice or
rats, it is preferable to use anti-antibodies formed from
sheep, which are directed against the Fc part of the
monoclonal antibody.
Further subject matter of the invention is a reagent for the
d~fferentiation of liver and bone isoenzymes of alkaline
phosphatase and for the specific determination of one of the
two isoenzymes plus the other isoenzyme in body fluids,
especially in serum or plasma, which contains a system for
the detection of alkaline phosphatase and a means for the
differentiation of the two isoenzymes, which is characterized
by containing as a means for the differentiation of the two iso-
enzymes a monoclonal antibody against one of the two enzymes,
which cross-reacts but slightly with the other isoenzyme.
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The reagent contains the monoclonal antibody preferably fixed
on a solid support. It can also contain the monoclonal
antibody in free form, and additionally an anti-antibody
directed against the monoclonal antibody.
The above statements on the process and on the monoclonal
antibody apply accordingly also to the reagent in accordance
with the invention.
The invention affords a simple and quick differentiation and
selective determination of either the liver or the bone
isoenzyme of alkaline phosphatase in addition to the other
isoenzyme in body fluids. The activity of the second
isoenzyme in the test fluid is found as the difference
between the total activity, which can be determined by
conventional methods, and the activity of either the liver or
the bone enzyme determined in accordance with the invention.
In the appended figures the following are represented:
Fig. 1 Extinction [mE] in an ELISA with the monoclonal
antibody B 4 - 50 as a function of the
concentration used, in [U/l] on liver (~O isoenzyme
and bone (a) isoenzyme of alkaline phosphatase.
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Fig. 2 Calibration curve for determining the content (%)
of liver isoenzyme of alkaline phosphatase in human
sera. P1 and P2 are serum samples (see Example 5).
The following examples explain the invention.
Example 1
A) Immunization of Balb/c mice with alkaline phosphatase
from human bone
Balb/c mice are immunized intraperitoneally with 100
mcgm of alkaline phosphatase from human bone in complete
Freund's adjuvant. At a rate of about every eight weeks
the immunization is continued with S0 mcgm of enzyme in
incomplete Freund's adjuvant. Four days before fusion
the final immuni~ation is performed intravenously with
50 mcgm o-f enzyme in physiological salt solution.
B) Fusion of the mouse spleen cells with myeloma cells
The fusion of spleen cells of Balbtc mice immunized in
accordance with A), with Ag8. ~ATCC CRL 1580) or
SPL/D (ATCC CRL 1581) myeloma cells is performed by the
standard me~hod in accorda~ce with W. B. Foster et al.,
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. ,
Thrombosis Research 28 (1982) pp. 649-661. The fusion
ratio of spleen cells to myeloma cells is 5 : 1. The
fusion products are disseminated on [size] 24 culture
dishes and fed with 5~104 peritoneal exudate cells per
culture dish. Positive primary cultures are cloned by
"limiting dilution" 3 to 4 weeks after fusion. The
cells are laid aside in [size] 96 culture plates and fed
with 2~104 peritoneal exudate cells. The resultant
clones are tested for their ability to produce antibody,
and positive clones arP stored either in liquid nitrogen
or used for the production of MAK in mouse ascites. In
this manner the hybridoma cell line ~4 - 50 can be
obtained, which has been filed in the American Type
Culture ~o].lection under the number ATCC 8571.
C) Production of the monoclonal antibody on mouse ascites
Female Balb/c mice are twice injected intraperitoneally
with 0.5 ml of Pristan 7 days apart. Each mouse is
inoculated intraperitoneally with about 5 10~
hybridoma cells obtained in accordance with B). After 8
to 14 days, ascites is withdrawn 1 to 3 times. It is
purified on an anion exchanger column and the pure
monoclonal antibody is obtained. By means of the
hybridoma cell line B 4 - 50, the monoclonal antibody B
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4 - 50 is obtained, which is directed against the liver
isoenzyme and has a cross-reactivity against the bone
isoenzyme of 20%. It belongs to the subclass IgG 1 K.
Example 2: Enzyme immunoessay (ELISA) on antibodies binding
alkaline phosphatase
In order to learn the presence and specificity of alkaline
phosphatase binding antibody in the serum of immunized mice
or in the supernatant liquid from the culture of the hybrid
cells or in ascites, an ~LISA is used as the test principle:
. ~
100 microliters of polyclonal rabbit antibody against mouse
antibody are placed in each depression in a "Dynatech
Microfluor" microtitration plate, in a concentration of 10
mcgm/ml for 18 hours at 4C in 0.05 M sodium carbonate buffer
pH = ~.3. Then it is coated over with 1~ cattle serum
albumin in 50 mM of tris buffer, pH = 7.1. 100 microliters
of sample per depression (serum, culture supernatant or
ascites, each in appropriate dllution) are incubated for 5 h
at room temperature. Then incubation is performed with 100
microliters of human bone alkaline phosphatase or with human
liver alkali~e phosphatase in a concentration of 100 units
per liter (determined with commercial reagent with
paranitrophenyl phosphate as substrate, 37C) in tris ~uffer,
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50 mM, pH = 7.1, for 4 h at room temperature. After thorough
washing with tris buffer, 50 mM, pH = 7.1, 0.9% NaCl, the
reagent 4-methylumbelliferyl phosphate of a concentration of
3O8 mM in 1.03 M diethanolamine buffer, pH = 10.4, with 0.5
mM MgC12 is added. The -fluorescence of the released 4-
methylumbelliferone is measured in a "Dynatech
Microfluorreader" after 30 to 60 minutes. The cross-
reactivity of the monoclonal anti~ody between bone and liver
alkaline phosphatase is calculated from the ratio of the
measured fluorescence intensities.
Example 3: Determination of the total content of alkaline
phosphatase in liquids, especially in human serum
3 ml of a commercial test reagent for alkaline phosphatase
(e.g., that of Boehringer Mannheim, Cat. Order No. 415,278)
is brought to a temperature of 37C in a photometer. After
the addition of 50 microliters of sample (serum or solutions
of alkaline phosphatases) the extinction increase at 405 nm
is determined as E/minute. By multiplying the reaction speed
by a factor of 3300, the total alkaline phosphatase
concentration of the sample is ohtained in units per liter.
Example 4~ Determination of the affinity of the liver and
bone isoenzymes of alkaline phosphatase for the
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monoclonal antibody B 4 - 50.
ELISA plates made by the firm of Nunc are coaked with 10
mcgm/ml of monoclonal antibody B 4 - 50 (100 mcl per
depression~ in 0.05 M sodium carbonate buffer, pH = 9.4, at
4C, for 18 hours. After overcoating (0.01 M sodium
phosphate buffer, pH = 7.4, 0.14 M sodium chloride, 1% cattle
serum albumin, 2 h, room temperature~ incubation is performed
for 2 h at 30C with human liver alkaline phosphatase in the
one case and with human bone alkaline phosphatase in the
other, in a concentration of 5 to 100 U/l (determined
according to Example 5) in 0.05 M tris buffer, pH = 7.1, 0.9%
sodium chloride. Then a thorough washing i5 performed (0.05
M tris buffer, pH = 7.1, 0.14 M sodium chloride, 0.05~ Tween-
20) and the plates, each depression with 100 microliters of a
suitable substrate solution for the alkaline phosphatase (for
example the commercial substrate paranitrophenyl phosphate,
Boehringer Mannheim Cat. Order 415 278), are incubated for 30
to 60 minutes (room temperature). The extinctions are then
evaluated with a "Dynatech Microelisareader". Figure 1 shows
the extinctions measured as a function of the amounts of
alkaline phosphate used. It can be seen that the monoclonal
antibody B 4 - 50 binds the bone alkaline phosphatase more
poorly than the liver alkaline phosphatase.
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ExamPle 5: Determination of human liver and bone isoenzym2s
of alkaline phosphatase in serum samples
"Dynatech Microfluor" microtitration plates are coated, half
with monoclonal antibody ~B 4 ~ 50) against human liver alkaline
phosphatase in accordance with the invention, and half with a
purified conventional unspecific monoclonal antibody (here
referred to as B 4 - 78) agalnst human alkaline phosphatase.
(100 mcl per depression), 10 mcgm/ml, 0.05 M sodium carbonate
buffer, pH = 9.5 4C, 18 h).
Then the plate ls thoroughly washed with buffer (0.01 M
sodium phosphate buffer, pH = 7.4, 0.14 M sodium chloride,
0.05% Tween-20, 0.02% NaN~). Pure human liver isoenzyme of
alkaline phosphatase (100~ L-AP), pure human alkaline
phosphatase from bone (0 ~ L-AP), and defined mixtures of the
two isoenzymes in the concentration range from 50 to 100 U/l
(determined in accordance with Example 3) are placed, in the
one case in depressions with the unspecific antibody, and in
the other case in depressions with the specific antibody.
The serum samples P1 and P2 with unknown alkaline phosphatase
content are diluted (with 0.05 M tris buffer, pH = 7.1, 0.9
NaCl) to a concentration of 50 to 100 U/l (determined
according to Example 5) and again distributed to depressions
containing specific antibody in the one case and unspecific
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antibody in the other. Incubation is performed for 2 h at
30C. Then a thorough washing is performed (0.05 M tris
buffer, pH = 7.4, 0.14 M NaCl, O.OS% Tween-20). Then to each
depression there is added 100 mcl of a 3.8 mM 4-
methylumbelliferyl phosphate solution in 1.03 M
diethanolamine buffer with O.S mM MgCla, pH _ 10.4. After 30
to 60 minutes the fluorescence of the 4-methylumbelli~erone
is measured in the "Dynatech Microfluorreader". The result
is the calibration curve given in Figure 2, when the
fluorescence of the (samplss) in the depressions
containing L-AP specific monoclonal antibody relative to the
fluorescence intensities with unspecific monoclonal antibody
are plotted against the percentage content of human liver
alkaline phosphatase of the standard solutions. From the
relativized fluorescence intensities of the samples it is
possible to read Erom the calibration curve the percentage
content of liver isoenzyme of alkaline phosphatase in the
sample ~P1 and P2 in Figure 2). From this percentage content
and the total alkaline phosphatase concentration ~determined
according to Example 5), the concentration of L-AP and K-AP
(bone alkaline phosphatase) in the samples can be ca]culated
in the unit U/l.
The sample P1 accordingly contains 29~ L-AP and 71~ K-AP;
sample P2 contains 85% L-AP and 15~ K-AP.
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It will be understood that the specification and
examples are illustrative but not limitative of the present
invention and that other embodiments within the spirit and
scope of the invention will suggest themselves to those
skilled in the art.
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