• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    1. Process for the specific determination of pancreatic alpha-amylase in body fluids, especially in serum, plasma, duodenal juice or urine, by reaction with a system for the detection of alpha-amylase in the presence of a monoclonal antibody which inhibits salivary alpha-amylase, characterised in that, as inhibitor, there is used a first monoclonal antibody which specifically inhibits the salivary enzyme by more than 70% but less than 97%, in combination with a second monoclonal anti-salivary alpha-amylase antibody which inhibits this enzyme by less than 10%.
    公开号:SU1637670A3
    申请号:SU4027915
    申请日:1986-07-18
    公开日:1991-03-23
    发明作者:Ленц Хельмут；Гербер Мартин；Альберт Винфрид
    申请人:Берингер Маннхайм Гмбх (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to a method for determining o (amylase of the pancreas along with o (amylase of saliva for diagnostic purposes).
    The aim of the invention is to improve the accuracy of determination of pancreatic 0 -amylase.
    The method is carried out as follows.
    The inhibitory efficacy of monoclonal antibodies, which do not fully satisfactorily suppress the enzyme of saliva, is synergistically enhanced by using only a binder, but practically not suppressing or incompletely suppressing monoclonal antibodies, and quantitative inhibition of the enzyme saliva can be achieved by obtaining 100 % activity of pancreatic enzyme. In this way, the incubation time is significantly reduced.
    For the proposed method, preferably a semi- as a first monoclonal antibody is used. Registered from .NCACC under the numbers (99D12) 84122003 and (89E2) 84122004 (National Collection of Animal Cell Cultures, Norton Down, UK) antibody cell cultures. As the second monoclonal antibody, preferably used are published in European Application No. 84114172.4 binding antibodies registered in the NCACC under the numbers 84111301 and
    about
    OE
    one
    OB 1

    about
    84111302 cell cultures (preferably NCACC No. 84111301).
    The required amount of first and second antibody for certain antibody preparations can be easily determined with a small number of preliminary experiments. Preferably, at least 5 μg / ml, böpee, preferably at least 20 μg / ml of the first monoclonal antibody and at least 1 μg / ml, more preferably at least 5 μg / ml of the second monoclonal antibody are used.
    Monoclonal antibodies used can be obtained by immunizing experimental animals with natural or modified C-amylase of saliva, by fusing B-lymphocytes of immunization animals thus obtained with transformable reagents, cloning and culturing the hybrid cells thus formed, which produce monoclonal antibodies, and by isolating the latter. Suitable animals for the production of saliva OC amylase antibodies are rats and mice.
    I
    Immunization is performed either with natural human saliva Ct-amylase or with modified saliva amylase. If a natural enzyme is used, standard, -electrophoretic homogeneous preparations are suitable for this. 1 preparations. Chemically modified saliva o / amylase can also be tormented in accordance with well-known truss modification methods. tone (published application of the Federal Republic of Germany N ° 254: 994). Suitable modifying agents IR.LYUGSYA, for example, N-bromointeramid (NBS) during the oxidation of cruciate: grimgofan in protein (BBA 143 (1967) 46L-472), carboxymethylation with ether and succinic acid of acid (1AA), which mainly affects the heptamethylamine with ester and succinic acid (1AA), which mainly affects the heptamethylation with ester and succinic acid (1AA), which mainly affects the heptamethylation with ester and succinic acid (1AA), which mainly affects the heptamethylation with ester and succinic acid (1AA), which mainly affects the heptamethylamine with ester and succinic acid (1AA), which mainly affects the acid (1AA), which mainly affects the ester and lactic acid (1AA). , nitriding with the granmer i -num (TNM) (J. Biul. Chem. 238 (1963) E07), and hacking all the way from the world (iropan sulphaniplopidium) and (Mi th. Enzymul. (25 / 1972)). In this case, it is the most appropriate of all tfptfpplnnyi using TNM fi rm ii, Mi ni hivib n unions of field (forest) mushrooms, i n t, - tesgvennyi f ".- mixed with n, -n i." mch zht
    HOUSE MICE
    five
    0 5
    0
    5 0 5 0 5
    Immunization is accomplished by simply administering a natural or modified enzyme in combination with a stimulant. Aluminum hydroxide is used as a stimulator in combination with a bacterium (the causative agent of whooping cough Bordeaux – Zhang). If natural immunization with salivary b-amylase in house mice or TNM-modified K-amylase in field (forest) mice is used for immunization, then immunization is carried out mainly for 9 months, using at least seven immunizations (injections of I.P. .).
    After immunization, the lymphocytes derived from the bone marrow of immunized animals are connected by conventional methods with transforming agents, for example myeloma cells, transforming viruses, for example Epstein-Barr virus or described in German laid out application No. 3245665. The compounding is carried out in accordance with known methods of Köhler and Milstein (Nature 256 (1975) 495-497). The hybrid cells thus formed are cloned in the usual manner, for example using standard cell rock, and the resulting clones, which form the desired monoclonal antibodies, are grown. Due to cancer-like Hybrid cell growth, the latter can continue to be cultured indefinitely and produce the desired monoclonal antibody in any amount.
    Fg1 of the proposed method of detection can use the monoclonal antibodies described above or fragments (fab fragments) having their respective immunological properties. Therefore, monoclonal antibodies are also understood in this case as fragments. Both the complex antibody and its fragments can be used in immobilized form.
    The determination of C-amylase is carried out in accordance with methods known for this purpose. Since the combination of monoclonal antibodies selectively inhibits salivary tin T-amylase and it thus allows you to determine the activity of fermesh, not causing damage to the enzyme of the peritoneal gland, obtained by determining d (-amylase in the presence of monoclonal antibodies
    Only pancreatic enzyme activity is appropriate.
    The proposed method is preferably implemented using a system for detecting o (.- amylase, which contains maltopolyosis with 4-7 glucose residues in the molecule, malto-phosphorylase, α-phosphophlogne, glucose-6-phosphate dehydrogenase, and diphosphopyridine nucleotide.
    Another preferred detection system (α-amylases consists of starch groups modified by detectable groups. The concept of modified starch includes, for example, starch that is modified by definition groups, for example, the standard product called Phadebas of Pharmacia (Sweden), or The product described in the German application No. 2812154 laid out, or the starch changed during the decomposition process, for example, carboxymethyl starch and limiting dextrans (all these systems are known).
    To implement the proposed method, the test liquid is either first incubated using the antibodies used in the method and then used in the usual amylase test, or added to the mixture of antibodies with the amylase detection reagent, and then after incubation, the determination is started by adding a substrate. The duration of the incubation period depends on the activity of the antibodies used and ranges from 0.5 to 10 minutes, in particular about 5 minutes.
    Since the separation of saliva 66-amylase is desirable, one of the monoclonal antibodies, both in complex form and in the form of fragments, may exist fixed on a solid carrier, for example, on immunosorption paper or on the surface of plastic tubes or flexible tubules. Thus, salivary type α-amylase is attached to the carrier (i.e., solid phase).
    Monoclonal antibodies that specifically suppress human saliva lasers up to a maximum of 93% are purified by precipitation with (NHy) SOy and using DEAE chromatography according to conventional methods. A monoclonal antibody that specifically binds human salivary α-amylase is also purified. Monoclonal antibody or monoclonal antibodies are mixed in solution with human amylase and this mixture is incubated in
    g for 5 min at room temperature. This mixture is then added to the amylase reagent and the amylase activity is measured. The control is amylase, which is mixed with
    5 with no monoclonal antibody buffer. Alternative monoclonal antibodies or monoclonal antibodies are added to the part of the amylase reagent, glucosidase solution. An amylase solution is then added and the mixture is incubated for 5 minutes. The reaction with the substrate, G-iPNP (P-nitrophenylmaltoheptaoside), is started. Glucoside solution is used as a control.
    5 without monoclonal antibodies
    Table 1 presents the results of the method of serum onset (mixed monoclonal antibody and amylase); suppression of human L-amylase slyu-t
    0 and pancreas using suppressive monoclonal antibodies NCACC 84122003 (MAK I) or 84122004- (MAK II) and / or the binding monoclonal antibody NCACC 84111301 (MAK III) (as a substrate of GyPNP, GA - general activity, A - activity with monoclonal antibodies, RA - residual activity).
    "From Table 1 it can be seen that the combination of a suppressive specific monoclonal antibody, as well as a specific human 0 (α-alaea monoclonal antibody) affects
    5 synergistic increase in suppression. This effect does not depend on the substrate. This is found in both the high molecular weight substrate (stained starch) and the short chained substrates, for example GyPNP (P - nitrophenyl maltopentaoside). The effect is also observed with the substrate. Salivary amylase is also suppressed in the human body synergistically using two monoclonal anti-5
    tel.
    Reagent for the specific determination of pancreatic Ot-amylase along with saliva-amylase in fluids
    system, in particular, serum, duodenal juice, plasma or urine, contains a system for detecting OC-amylase and a monoclonal antibody against cC-amylase of saliva, which is different in that it contains the first monoclonal antibody that suppresses the saliva enzyme less than up to 97%, and is used in combination with a second monoclonal antibody against saliva α-amylase, which suppresses this enzyme to less than 10%. Thus, the proposed reagent contains a system for the detection of (amylases.
    The invention facilitates a simple, rapid and accurate determination of pancreatic β-amylase (h-PA along with salivary type ut-amylase (h-SA) in body fluids and thereby significantly improves clinical diagnostic capabilities.
    Example 1. Suppression of human pancreatic K-amylase and saliva using the first MAC (NCACC 84122004) and in combination with the second MAC (NCAAC 84111301); serum onset method.
    Reagent (buffer) 100 ml PO and 150 ml NaCl, 6% of cattle serum albumin, pH 7.1.
    Amylase reagent: GfPNP (Böhringer, Mannheim, order number from the catalog 568569, 37 ° C).
    MAC 1: 0.63 mg / ml of the first MAC in the buffer.
    MAC 2: 0.63 mg / ml of the first MAC and 0.105 mg / ml of the second MAC in buffer.
    MAC 3: 0.105 mg / ml second MAC in buffer.
    In this case, h-SA - 3000 urea / l (GTPNP) in the buffer, h-PA - 3000 urea / l () in the buffer.
    100 μl of human saliva or pancreatic ob-amylase are mixed with 100 μl of buffer or various monoclonal antibody solutions and incubated for 5 minutes at room temperature. Then 25 µl of these mixtures add 10–1000 µl of amylase reagent maintained at a constant temperature of 37 ° C, and the amylase activity is determined in accordance with the instructions. The residual activity is calculated by the formula-RA,% Activity with MAX.
    w.-.-. ------. ---- .--. f--, 1 Y9
    . i Activity without MAC
    jg j5
    20
    25
    thirty
    "
    40
    45
    -Q
    five
    The corresponding residual activity of human <RTI ID = 0.0> α / -amylase </ RTI> saliva is 8.6% with the first MAK, 96.9% with the second MAA and 2.7% with both MAC, the residual human 0 activity (.- pancreatic amylase, respectively, 1, 99.6 and 99.7%.
    Example 2. Suppression of amylase using the first MAC (NCACC 84122003) and / or the second MAC (NCACC 84111301), a variant of serum onset.
    The reagents are similar to those used in Example 1, only MAC 1 and MAC 2 are modified (
    MAC 1: 0.525 mg / ml perv9 MAC in buffer,
    MAC 0.525 mg / ml of the first MAC and 0.105 mg / ml of the second MAC in the buffer.
    The experiment was carried out analogously to example 1,
    The residual activities of human saliva tf-amylase are 9.1% with the first MAC, with 96.9% with the second MAC, and with both MAK 2.5%, ocTatO4m, i.e., the activity of human pancreatic 0-amylase respectively 99.9, 99 , 6 and 99.7%.
    Example 3. Suppression of human OS-amylase of saliva and pancreas using the first MAC (NCACC 84122004) with or without the second MAC (NCACC 84111301), the substrate-based method.
    Reagents: buffer, human 6 (- saliva amylase (h-SA) and human
    -amylase pancreas (h-PA) as in example 1.
    MAC 1H: first MAC in buffer, different concentrations.
    MAC 2H: second MAC in buffer 0.513 mg / ml.
    At the same time, K-glucosidase from amylase reagent (Weringer, Mannheim, order number 568569), substrate from amylase reagent.
    To 880 µl of W-glucoeidase, 100 µl of MAK 1 solution are added, as well as 10 µl of MAK 2y solution or 10 µl of buffer. 25 μl of human saliva or pancreatic human {/ -amylase is mixed into this mixture. This mixture is incubated at 37 ° C for 5 minutes. Then, by the addition of 100 µl of the substrate solution, the onset of amylase activity is initiated and carried out in accordance with the instructions.
    The control is a mixture of II-glucosidase with 20 μl of buffer, as well as the corresponding amylase. Activity with monoclonal antibodies, divided by activity without monoclonal antibodies, gives residual activity (in percent).
    The results of the experiments for various concentrations are summarized in Table 2.
    The data of Table 2 indicate that the concentrations of antibodies used make it possible with high accuracy to determine the pancreatic GC 15 amylase (by the known method only
    权利要求:
    Claims (1)
    [1]
    90%). Invention Formula
    Method for determination of pancreatic VS-amylase in biological 20
    liquids, which include the addition of monoclonal antibodies against tf amlase of saliva to the test material, followed by registration of pancreatic C-amylase, characterized in that, in order to improve the accuracy of the method, antibodies of CASS strains No. 84122003 are used as monoclonal antibodies or CACE V 84122004 at a concentration of 5-20 µg / ml with saliva α-amylase binding activity of more than 97%, then further added second monoclonal antibodies against saliva tf-amylase of CASS strains No. 84111301 or No. 84111302 in equal volume and concentration 1 - 5 µg / ml with enzyme binding activity greater than 10%; the mixture is incubated for 0.5-10 minutes.
    Indicators . MAC I
    IAC I IAC III IAC III IAC I / Ill IAC I / Ill
    MAK - concentration in the experiment, mg / l
    20
    Table I
    table 2
    5/1
    20/5
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    引用文献:
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    DE2543994C3|1975-10-02|1979-10-11|Behringwerke Ag, 3550 Marburg|Protein-modified compounds of pregnancy-specific β, -glycoprotein and their use as a component of immunizing agents|
    DE2812154C3|1978-03-20|1980-09-11|Boehringer Mannheim Gmbh, 6800 Mannheim|Method for the determination of α-amylase|
    JPS6111593B2|1981-11-16|1986-04-03|Fuji Rebio Kk|
    DE3245665A1|1982-05-04|1983-11-10|Boehringer Mannheim Gmbh, 6800 Mannheim|METHOD FOR PRODUCING PERMANENT ANIMAL AND HUMAN CELL LINES AND THE USE THEREOF|
    US4514505A|1982-05-21|1985-04-30|The Trustees Of Columbia University In The City Of New York|Monoclonal antibody mixtures and use thereof for enhanced sensitivity immunoassays|
    DE3323245A1|1983-06-28|1985-01-10|Merck Patent Gmbh, 6100 Darmstadt|METHOD AND REAGENT FOR DETERMINING AMYLASE|
    DE3342736A1|1983-11-25|1985-06-05|Boehringer Mannheim Gmbh, 6800 Mannheim|HYBRIDOMA ANTIBODY|
    DE3404876A1|1984-02-11|1985-09-05|Gödecke AG, 1000 Berlin|METHOD FOR IMPROVING THE SELECTIVE ACTIVITY INHIBITION OF HUMAN PANCREAS AND SPOKEL AMYLASE, AND AN INHIBITOR PREPARATION SUITABLE FOR THIS|
    DE3500526A1|1985-01-09|1986-07-10|Boehringer Mannheim Gmbh, 6800 Mannheim|SPECIFICALLY INHIBITING S-AMYLASE MAK|DE3621271A1|1986-06-25|1988-01-07|Boehringer Mannheim Gmbh|METHOD AND REAGENT FOR DETERMINING ALPHA AMYLASE|
    DE3903114A1|1989-02-02|1990-08-09|Boehringer Mannheim Gmbh|METHOD FOR DETERMINING AN ENZYME FROM AN ISOZYME MIXTURE, AND TEST CARRIER SUITABLE FOR THIS, AND USE THEREOF|
    DE3929355A1|1989-09-04|1991-03-07|Boehringer Mannheim Gmbh|PROCESS FOR SPECIFIC DETERMINATION OF PANCREASAMYLASE|
    JPH085919B2|1989-11-10|1996-01-24|日本商事株式会社|Saliva-α-amylase activity-inhibiting monoclonal antibody and pancreas-α-amylase fractional quantification method using the same|
    JPH04106694U|1991-02-26|1992-09-14|
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    法律状态:
    2005-01-10| REG| Reference to a code of a succession state|Ref country code: RU Ref legal event code: MM4A Effective date: 20020719 |
    优先权:
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