CA1293209C - Method and reagent for the differentiated determination of isoenzymes of alkaline phosphatase and monoclonal antibody suitable for this purpose - Google Patents
Method and reagent for the differentiated determination of isoenzymes of alkaline phosphatase and monoclonal antibody suitable for this purposeInfo
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- CA1293209C CA1293209C CA000484343A CA484343A CA1293209C CA 1293209 C CA1293209 C CA 1293209C CA 000484343 A CA000484343 A CA 000484343A CA 484343 A CA484343 A CA 484343A CA 1293209 C CA1293209 C CA 1293209C
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- isoenzyme
- alkaline phosphatase
- bone
- liver
- monoclonal antibody
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Abstract
Abstract The subject of the invention is a method and a reagent for the differentiation of the liver isoenzyme and bone isoenzyme of alkaline phosphatase and selective determination of the differentiated isoenzymes by measurement of the alkaline phosphatase activity, the differentiation of the two isoenzymes being performed by means of a monoclonal antibody which is directed against one of the two isoenzymes and has only a slight cross-reactivity with the other isoenzyme. The application furthermore relates to the monoclonal antibody suitable for the differentiation of the two isoenzymes.
These are obtained by immunization of experimental animals with pre-purified alkaline phosphatase from liver or bone, fusion of B-lymphocytes, cloning and cultivation of the hybridoma cells thus formed, and isolation of the monoclonal antibody from the latter.
These are obtained by immunization of experimental animals with pre-purified alkaline phosphatase from liver or bone, fusion of B-lymphocytes, cloning and cultivation of the hybridoma cells thus formed, and isolation of the monoclonal antibody from the latter.
Description
~Z~32~9 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 ' ~
~3~
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.
. ~ .
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 3~0~
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 `` ~Z93209 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 1~3;~:09 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 3;~09 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 ~29~g 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 ,~
3~9 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 Z~32~)9 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 ~ Z~3209 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.
`'``` ~z~zo9 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.
93Z~)~
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., 12~3~2~)9 . , 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
, .
~L~93Z09 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, ~ ~r~d~ ~f~< 14 ~93~g 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 raC~ f k 15 93ZO~
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.
-~ ~z~3ZOg 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 ~ 93Z~)9 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.
~3~)9 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.
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 ' ~
~3~
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.
. ~ .
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 3~0~
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 `` ~Z93209 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 1~3;~:09 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 3;~09 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 ~29~g 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 ,~
3~9 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 Z~32~)9 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 ~ Z~3209 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.
`'``` ~z~zo9 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.
93Z~)~
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., 12~3~2~)9 . , 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
, .
~L~93Z09 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, ~ ~r~d~ ~f~< 14 ~93~g 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 raC~ f k 15 93ZO~
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.
-~ ~z~3ZOg 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 ~ 93Z~)9 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.
~3~)9 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.
Claims (14)
1. A method for the separation of liver isoenzyme and bone isoenzyme of alkaline phosphatase in a sample containing a mixture thereof for selective determination of the differentiated iso-enzymes, by measuring the alkaline phosphatase activity, comprising the steps of reacting the mixture with a monoclonal antibody which is directed against the liver isoenzyme, and shows a cross-reactivity of 20% or less with the other isoenzyme, to form an isoenzyme-antibody complex, and separating the isoenzyme-antibody complex from the unconjugated isoenzyme, whereby the isoenzymes are separated.
2. The method of claim 1, wherein said mono-clonal antibody directed against alkaline phosphatase is obtained by immunization of laboratory animals selected from rats and mice with pre-purified alka-line phosphatase from bone, fusion of B-lymphocytes of the immunized animals with transforming agents to form hybrid cells, cloning and cultivation of the hybrid cells, and isolation of the monoclonal anti-body from the cultivated hybrid cells.
3. The method of claim 2, wherein immunization is performed with aluminum hydroxide and Bordatella pertussis or Freund's adjuvant, as adjuvant.
4. The method of claim 2, wherein myeleoma cells are used as transforming agents.
5. A reagent for the differentiation of the liver isoenzyme and bone isoenzyme of alkaline phosphatase and for the specific determination of one of the two isoenzymes comprising:
an agent for the detection of alkaline phosphatase; and a means for the differentiation of the two isoenzymes, said means for the differentiation of the two isoenzymes containing a monoclonal antibody against liver isoenzymer which cross-reacts 20% or less with the bone isoenzyme.
an agent for the detection of alkaline phosphatase; and a means for the differentiation of the two isoenzymes, said means for the differentiation of the two isoenzymes containing a monoclonal antibody against liver isoenzymer which cross-reacts 20% or less with the bone isoenzyme.
6. A monoclonal antibody characterized in that it reacts specifically with liver isoenzyme of alkaline phosphatase and cross-reacts 20% or less with bone isoenzyme of alkaline phosphatase.
7. A method of preparing the monoclonal antibody of claim 6, comprising the steps of immuniz-ing laboratory animals selected from rats and mice with pre-purified alkaline phosphatase from bone, fusing the B-lymphocytes of the immunized animals with transforming agents to form hybrid cells, cloning and cultivating the hybrid cells, and isolat-ing the monoclonal antibody from the cultivated hybrid cells.
8. The method of claim 7, wherein immunization is performed with aluminum hydroxide and Bordatella pertussis of Freund's adjuvant, as adjuvant.
9. The method of claim 7, wherein myeloma cells are used as transforming agents.
10. A method for the separation of liver isoenzyme and bone isoenzyme of alkaline phosphatase in a sample containing a mixture thereof for selective determination of said isoenzymes comprising reacting a mixture containing both liver and bone isoenzymes with a monoclonal antibody which is directed against the liver isoenzyme, and shows a cross-reactivity of 20% or less with the bone iso-enzyme under conditions favourable to formation of an isoenzyme-antibody complex, and separating the isoenzyme-antibody complex from the unconjugated isoenzyme.
11. A monoclonal antibody which specifically binds to an isoenzyme of alkaline phosphatase, characterized in that it reacts specifically with liver alkaline phosphatase isoenzyme, and shows a cross-reactivity of 20% or less with the bone iso-enzyme.
12. A hybridoma cell line which produces monoclonal antibodies directed against the liver isoenzyme of alkaline phosphatase, and shows a cross-reactivity of 20% or less with the bone iso-enzyme, obtainable by fusion of B-lymphocytes of rats or mice immunized with the bone isoenzyme, with a transforming agent.
13. A cell line according to claim 12, in which the transforming agent comprises myeloma cells.
14. A method for formation of monoclonal antibodies directed against the alkaline phosphatase liver isoenzyme, and showing a cross-reactivity of 20% or less with the bone isoenzyme, comprising immunization of a murine specimen with pre-purified alkaline phosphatase from bone, fusion of B-lympho-cytes of the specimen with transforming agents to form immortal cells, cloning and cultivation of the cells and isolation of the monoclonal antibody therefrom.
#17-12/28/1990
#17-12/28/1990
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000484343A CA1293209C (en) | 1985-06-18 | 1985-06-18 | Method and reagent for the differentiated determination of isoenzymes of alkaline phosphatase and monoclonal antibody suitable for this purpose |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000484343A CA1293209C (en) | 1985-06-18 | 1985-06-18 | Method and reagent for the differentiated determination of isoenzymes of alkaline phosphatase and monoclonal antibody suitable for this purpose |
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| CA1293209C true CA1293209C (en) | 1991-12-17 |
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| CA000484343A Expired - Lifetime CA1293209C (en) | 1985-06-18 | 1985-06-18 | Method and reagent for the differentiated determination of isoenzymes of alkaline phosphatase and monoclonal antibody suitable for this purpose |
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1985
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