JPH044880B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antibodies against proteins. More specifically, the present invention relates to monoclonal antibodies against human prolyl hydroxylase using hybridomas. Prolyl hydroxylase is one of a group of enzymes involved in collagen biosynthesis, collagen metabolites and metabolism by enhancing turnover. This enzyme is said to be involved in the overproduction of collagen in blood vessel walls in arteriosclerosis and the overproduction of fibrous protein in liver cirrhosis. Arteriosclerosis, the extent of which cannot be predicted unless it causes serious complications such as stroke or myocardial infarction,
Alternatively, GOT (glutamine ogizaloacetate transaminase), GPT (glutamine pyruvate transaminase), LDH (lactate dehydrogenase), and γ-GTP (γ-glutamyl transpeptidase) activity measurements, which are used as conventional liver function determination methods, Measuring the amount of this enzyme in the blood has important significance for the diagnosis and prognosis of liver cirrhosis, which cannot be predicted and relies on biopsy. Radioimmunoassay and enzyme immunoassay have been considered as highly sensitive methods for measuring the amount of this enzyme in blood. Conventionally, anti-prolyl hydroxylase antibodies used in such immunoassays have been prepared from rabbits and other animals. It is obtained by immunizing animals with this enzyme. However, the antibodies (specific immunoglobulins) obtained in this way are mixtures with other immunoglobulins, and even if antibodies with high affinity for this enzyme are obtained, they are difficult to purify and are subject to quantitative limitations, making immunoassays difficult. When applied to a drug, there is a risk of variation in specificity and potency between lots. In most cases, as the purity of an antibody increases, so does its value as a specific antibody. For this reason, antibody-producing B cells or their precursor cells are fused with myeloma cells (myeloma cells) capable of continuous division and proliferation, resulting in a homogeneous fused cell (hybridoma) that has the characteristics of both these cells. Attempts have been made to obtain clones of the same, and it has been shown that clones thereof can be obtained (Ko¨hler et al.
Nature, 256, 495-497, 1975). Furthermore, monoclonal antibodies against malignant tumor antibodies and other various antigens have been produced using similar techniques (see Patent Publication No. 45407/1983). However, for human prolyl hydroxylase, monoclonal antibodies with uniform and highly specific cross-reactivity that are of practical utility have not been provided. The present invention is based on hybridomas.
The present invention provides an IgG class anti-prolyl hydroxylase antibody. That is, the present invention involves immunizing an animal with human prolyl hydroxylase, forming a hybridoma using anti-human prolyl hydroxylase antibody-producing cells and myeloma cells from the animal, cloning the hybridoma, and then immunizing it with prolyl hydroxylase. obtained by selecting and culturing clones that produce anti-prolyl hydroxylase antibodies that are reactive with
An anti-prolyl hydroxylase antibody of the IgG class, which has immunocross-reactivity with ratprolyl hydroxylase and has immuno-cross-reactivity with antigenic determinants present in human prolyl hydroxylase. Monoclonal antibodies are provided. The novel anti-human prolyl hydroxylase monoclonal antibody according to the present invention is extremely useful in the diagnosis and prognosis of liver cirrhosis. In other words, as liver cirrhosis progresses, the amount of human prolyl hydroxylase increases in the blood, but if it is possible to accurately measure the increase or decrease in the amount of human prolyl hydroxylase in the blood, it will be possible to diagnose liver cirrhosis or determine the treatment status. It is possible to judge accurately. Furthermore, the IgG class monoclonal antibody according to the present invention has the following advantages over other IgA class or
They have characteristic advantages that are significantly different from monoclonal antibodies of the IgM class. In other words, monoclonal antibodies of the IgA class and IgM class have strong nonspecific binding and react with proteins other than so-called human prolyl hydroxylase, so these monoclonal antibodies cannot be used in immunoassays. However, in this regard, the IgG class monoclonal antibody according to the present invention is useful for diagnosing liver cirrhosis or determining the treatment status. It has a particularly remarkable effect on the measurement of pitoprolyl hydroxylase. Furthermore, in research and development of drugs for the treatment of liver cirrhosis, a poisonous substance is usually administered to rats to artificially cause liver cirrhosis, and a therapeutic test substance is administered to the rats, and the amount increases or decreases as the drug is administered. By measuring the amount of latprolyl hydroxylase in the blood, the presence or absence of a therapeutic effect of the test substance is determined. In this case, the monoclonal antibody according to the present invention Since it has immunoreactivity with ratprolyl hydroxylase, it provides extremely excellent measurement effects when measuring ratprolyl hydroxylase in blood in model experiments using rats. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 (a) Antigen-prolyl hydroxylase (EC1,
Preparation of 14, 11, 2) Eur.J.Biochem. 52 using human placenta as material.
According to the method of Tuderman et al. described in 9-16 (1975), human prolyl hydroxylase was captured using affinity chromatography in which PolyL-Proline was coupled to Sepharose 4B with CNBr.
It was further purified and purified using a Bio-GelA-1.5m (Bio-Rad) column. The obtained enzyme preparation was sent to J.
Its purity was examined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel according to the method of Baum et al. described in Virol. 10 , 211-219 (1972) and found to be about 90%. (b) Preparation of antibody-producing cells First, three 8-week-old Balb/c female mice were
Initial immunization with human prolyl hydroxylase in complete Freund's adjuvant. Mice each receive 50 μg of human prolyl hydroxylase intraperitoneally in a 0.5 ml solution. moreover
On the 30th and 60th day, boost with the same amount of human prolyl hydroxylase dissolved in physiological saline. Intravenous administration (50μ
g/10μ physiological saline), and
After a day, sacrifice the mice, remove the spleen, and prepare splenocytes. (c) Preparation of myeloma cells The myeloma cells used in the method of the present invention are not particularly limited, and many animal cell lines such as mouse, rat, rabbit, and human can be used. Preferably, the cell line used is drug resistant so that unfused myeloma cells cannot survive in the selective medium, but hybridomas can survive. The most commonly used cell lines are 8-azaguanine-resistant and hypoxanthine-resistant.
It lacks guanine phosphoribosyltransferase (HGPRT) and cannot grow on hypoxanthine-aminopterin-thymidine (HAT) medium. In addition, some myeloma cell lines synthesize H chains and L chains, and when such cell lines are used, hybrid antibody molecules can be created that mix with the H chains and L chains synthesized by antibody-producing cells. It is desirable to use one that does not synthesize H chains or L chains. For example, P3-NS1-1- from the mouse myeloma MOPC-21 line.
Ag4−1, P3−X63−Ag8−U1, P3X63−
Ag8.6.5.3, SP2/0-Ag14, FO, SI94/
5XXO and BU1 can be suitably used. Although the P3-NS1-1-Ag4-1 strain was used here, care was taken not to increase the number of pre-fusion cells to a concentration higher than 2 x ¹⁰⁵ to 3 x ¹⁰⁵ cells/ml, and to maintain the viable cell rate as much as possible. I raised it. (d) Cell fusion using the following materials and methods. PPMI1640
The medium was RPMINo.1640 (Difco Laboratories) containing sodium bicarbonate (12mM), sodium pyruvate (1mM), L-glutamine (2mM),
Add potassium penicillin G (50μ/ml), streptomycin sulfate (50μg/ml) and amikacin sulfate (100μg/ml) and place on dry ice.
Adjust the pH to 7.2 and sterilize with a 0.2 μm Toyo membrane filter. NS-1 medium was prepared by adding sterilized fetal calf serum (Granite Diagnostic, Inc.) to RPMI-1640 medium supplemented as above.
A concentration of 15% (v/v) was added. HAT
The medium is NS-1 medium plus hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine (16 μM). HT medium has the same composition as HAT medium except that aminopterin is removed. polyethylene glycol 4000
(PEG4000, Merck) 50% in RPMI1640 medium
Prepare a (w/w) serum-free solution. 8-Azaguanine-resistant myeloma cell NS-1
The fusion with (P3-NS1-1-Ag4-1) is
Selected Method in Cellular Immunology
(ed.BBMishell and SMShiigi), W.H.
The method of Oi et al., described in Freeman and Company (1980), 351-372, was followed with some modifications. In this fusion, 1.5 × 10 ⁸ nucleated spleen cells (viable cell rate
95%) are fused with 2.8 x 10 ⁷ myeloma cells NS-1 (95% viable cell rate). The spleen cells and bone marrow cells are washed with serum-free RPMI-1640 medium. Next, suspend in the same medium and mix in the above ratio for fusion. Conical styrene resin test tube with a capacity of 50 ml (Corning Glass
40 ml of serum-free RPMI-
Centrifuge at 400 xg for 10 minutes through 1640 medium, and aspirate the supernatant completely. Precipitate cells at 37℃ for 50%
1 ml of PEG4000 solution with gentle stirring
Add the solution dropwise for 1 minute, and stir for an additional 1 minute to resuspend and disperse the cells. Next, 37℃ heated RPMI−
Drop 1 ml of 1640 medium over 1 minute. After repeating this operation once more, 7 ml of the same medium is added dropwise over 2-3 minutes with constant stirring to disperse the cells. This is centrifuged at 400 xg for 10 minutes, and the supernatant is completely removed by suction. Next, to this precipitated cell
Immediately add 10 ml of 37℃ heated NS-1 medium,
Disperse large clumps of cells by carefully pipetting with a 1 ml pipette. Furthermore, 20 ml of the same medium is added to dilute it, and 5.9×10 ⁵ cells/0.1 ml are seeded per well into a polystyrene 96-well microwell (Corning Glass Works). As a pretreatment for the 96-well microwell used at this time, 0.2 ml of NS-1 medium was added, kept overnight in a carbon dioxide gas incubator (37°C), and the medium was removed by suction before use. Microwells with completed cell fusion in 7% carbon dioxide gas/93% air temperature 37
Incubate at 100% humidity. (e) Selective proliferation of hybridomas using selective medium On the first day of culture, add 2 drops (approximately 0.1 ml) of HAT medium using a Pasteur pipette. 2, 3, 5, 8,
On day 11, replace half of the medium (0.1 ml) with fresh HAT medium. Switch to HT medium on day 14 and repeat the same operation every 3-4 days. Usually 2-3
Sufficient hybridoma growth was observed within a week (fusion rate 83%). All wells in which hybridomas are grown are checked for positive wells by the solid phase-antibody binding test described in the next section. Next, using each detected positive well (20/288 wells) as a feeder, 1 ml of HT medium containing ¹⁰⁷ mouse thymocytes was added to a polystyrene 24-well cell well (Corning Glass Works), and each of the detected positive hybridomas was Transfer the entire contents of 20 pieces.
This is similarly incubated at 37° C. for about one week in the presence of 7% carbon dioxide. During this time, replace 0.5 ml of supernatant of each well with 0.5 ml of fresh HT medium once or twice. When the hybridomas have grown sufficiently, positive results are reconfirmed using a solid phase-antibody binding test, and each is cloned using the limiting dilution method. In addition, the remaining liquid after use for cloning should be placed in a polystyrene ^25cm2 tissue culture flask (Cornin
Glass Works) to prepare samples for cryopreservation. (f) Search for anti-prolyl hydroxylase antibody-producing hybridomas by solid phase-antibody binding test. This method was published in Anal.Biochem. 104 , 205-214 (1980).
This is a slightly modified version of the method of Rennard et al. described in , and is suitable for detecting hybridoma antibodies.
96-well microtitration plate (Flow
Laboratories, Inc.) with 0.5-1.0 μg of prolyl hydroxylase, and further
Block with 1.0% bovine serum albumin (BSA). A portion of the supernatant of the hybridoma growth well is added to this and incubated at room temperature for about 1 hour. Horseradish peroxidase-labeled goat anti-mouse IgG (TAGO, Inc.) is added as a secondary antibody, and further incubated at room temperature for about 1 hour.
Next, hydrogen peroxide and the substrate O-phenylenediamine are added, and the degree of brown color produced is determined qualitatively with the naked eye or with a Corona two-wavelength microplate photometer (MTP-22, Corona Electric Co., Ltd.). Quantitated. (g) Cloning Since there is a possibility that two or more types of hybridomas are growing in each well, cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. NS-1 medium
Prepare a cloning medium containing 10 ⁷ mouse thymocytes as feeders per ml and add 5, 1 and 0.5 hybridomas per well to 36, 36 and 24 wells of a 96-well microwell.
Approximately 0.1 ml of NS-1 medium is added on days 5 and 12. Sufficient hybridoma growth is observed 14 to 15 days after the start of cloning, and a solid phase-antibody binding test is performed on groups in which 50% or more of the wells are negative for colony formation. If all wells tested are not positive, check the number of colonies in the antibody-positive wells and divide 4-6 wells with 1 colony in each well.
Select and re-clon. Finally, 8 clones were obtained. (h) In-vitro and in-vivo proliferation of single antibodies For single antibodies, the obtained clones are cultured (in-vitro propagated) in a suitable culture medium such as NS-1 medium,
It can be obtained from the culture supernatant (single antibody protein concentration is 10-100 μg/ml). On the other hand, in order to obtain antibodies in large quantities, use an animal of the same gene as the source of splenocytes and myeloma cells (Balb/c mouse).
The tumorigenesis promoter pristane (2,6,10,14
-tetramethylpentadecane, Aldrich
Chemical Campany, Inc.) per mouse.
Administer 0.5ml intraperitoneally. By similarly administering 1×10 ⁷ hybridomas intraperitoneally after 1 to 3 weeks, ascites with a single antibody protein concentration of 4 to 7 mg/ml can be obtained in vivo after 1 to 2 weeks. (i) Heavy chain and light chain isotypes of a single anti-prolyl hydroxylase antibody The obtained ascites was first transferred to a prolyl hydroxylase-coated microtitration plate as described above for the solid phase-antibody binding test. be combined with After washing, isotype-specific rabbit anti-mouse Ig antibody (Zymed
Laboratories). After washing, horseradish peroxidase-labeled goat anti-rabbit IgG (H+
L) Add antibody and use 2,2'-azino- as substrate
Detection was performed using di(3-ethylbenzothiazoline sulfate-6) and hydrogen peroxide. The results are summarized in Table 1. Of the antibodies examined, four were immunoconjugated with globulin chains γ ₁ /κ, and one with γ ₂ b/κ.
κ, two had α/κ and one had μ/κ. Example 2 Purification of single antibody and antibody titer Although ascites contains antibodies derived from host animals unrelated to prolyl hydroxylase, they are diluted tens of thousands to hundreds of thousands of times during enzyme immunoassay. There is no problem at all when using it.
However, if necessary, ascitic fluid is fractionated with ammonium sulfate (40% saturation).
After that, DEAE−Sephacel (Pharmacia Fine
Ion-exchange column chromatography method using Protein A-Sepharose CL-
4B (Pharmacia Fine Chemicals) affinity chromatography alone or
SephacrylS−300Superfine (Pharmacia Fine
It can be purified by a combination of gel filtration methods. Of the antibodies obtained, IgG ₁
Class: 40mM phosphate buffer containing 0.06M salt;
DEAE-Sephacel equilibrated at PH8.0 (2 x 38 cm)
It was obtained in the non-adsorbed fraction of the column and could be separated from the mouse-derived protein adsorbed on the column. This IgG ₁
The fractions were further equilibrated with SephacrylS-, which was equilibrated with 50mM phosphate buffer containing 0.42M NaCl, pH 7.4.
Gel filtration was performed on a 300 Superfine column (1.5 x 93.5 cm) (Figure 1). Also, the IgG _2b class is Immunochem.
Protein A-Sepharose CL-4B column (1.6×
3 cm). IgG _2b as shown in Figure 2
was eluted with 50mM acetate buffer, pH 4.3. The purity of the IgG ₁ and IgG _2b classes purified in this way was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the molecular weight was approximately 52K (H chain).
Two main bands of approximately 25K and 25K (L chain) were detected. For IgM and A class purification, add DEAE-Sephacel equilibration buffer to 0.06M NaCl.
Purification was performed under the same conditions as for the IgG ₁ class, except that 40mM phosphate buffer containing pH 8.0 was used and elution was performed with a gradient from 0.06M to 0.5M NaCl. The purity of the IgM class eluted in the Void Volume was examined by gel electrophoresis in the same manner as the IgG class, and the molecular weight was approximately 80k (H chain) and approximately 25k.
Two main bands (L chain) were detected.
For IgA, the molecular weight is approximately 64k (H chain) and approximately 25k (L chain).
Two main bands were detected. The titer of the single antibody purified as described above was measured by a solid phase-antibody binding test. For the IgA class enzyme-labeled second antibody, peroxidase-labeled goat anti-mouse IgA (Zymed
Laboratories Inc.) and peroxidase-labeled goat anti-mouse IgO for those of the IgM class.
(Zymed Laboratories Inc.) was used. Figure 3 shows the results of measuring the brown color produced in each well of a 96-well microtitration plate using a corona two-wavelength microplate photometer MTP-22.
IgG class clone numbers 3-2B12, 2-1C2,
3-3H9 and 3-6H5 had a strong binding ability with immobilized antigen, and the detection limit was an antibody amount of 0.1 to 0.2 ng, but in 2-5G8, the measured antibody amount was 0.5 to 0.2 ng.
The binding ability was weak in the 500ng range. On the other hand, for the IgA and IgM classes, the binding ability was strongest in the order of clone numbers 2-6G9, 2-7F8, and 3-4H2. Especially for 3-4H2 (IgM), the binding increased significantly on the high concentration side. Note that the detection limit for these was an antibody amount of 0.5 to 1.0 ng. Of the eight types of monoclonal antibodies obtained, except for clone numbers 2-1C2 and 2-6G9, they also reacted with rat fetal (internally removed) prolyl hydroxylase in the same manner as human prolyl hydroxylase (No. (Table 2). Example 3 Inhibition of the activity of a single anti-prolyl hydroxylase antibody Anti-human prolyl hydroxylase activity of a purified single antibody was determined according to the method of Hutton et al. as described in Anal Biochem. 16 , 384-394 (1966). 4-L-proline from chicken fetal protocollagen was used as a substrate.
Using ^3H -labeled water, the generated ^3H -labeled water ( ^3HHO ) is captured by vacuum distillation. Add Bray's reagent to a certain amount of this water and measure the radioactivity using a Packard Tri-Carb scintillation counter. That is, a predetermined amount of each single antibody was added to 0.2 μg of prolyl hydroxylase per test tube and pre-incubated at 30°C for 15 minutes, then the above substrate was added and further incubated at 30°C for 30 minutes. did.
Among the antibodies tested, only clone number 2-1C2 showed anti-prolyl hydroxylase activity in the IgG class. On the other hand, both the IgA and M classes showed anti-prolyl hydroxylase activity, but the degree of activity was greater in the IgA class than in the IgM class (Table 3). The above experiment investigated the reactivity of the single antibody obtained with the antigenic determinant of the prolyl hydroxylase active center, and for example, the activity of 2-1C2 was not completely inhibited. . However, no activity of this enzyme-antibody complex was observed in the supernatant obtained by further adding goat anti-mouse IgG and centrifuging it. Example 4 Detection of prolyl hydroxylase in placenta by indirect fluorescence antibody method Placenta was frozen in acetone-dry ice, cut into 4μ thick pieces using a cryostat, and cut into 4μ thick pieces with cold acetone.
After fixation for a minute, a 1:100 dilution of each ascites fluid was added to the sections and allowed to react at room temperature for 1 hour. Next, after washing with phosphate buffer (PBS), pH 7.2, the fluorescent reagent-labeled second
Fluorescein isothiocyanate-labeled goat anti-mouse IgG, IgA or IgM (1:
50 dilution, Miles and Zymed Laboratories Inc.)
reacted. After washing, the sections were mounted in 50% glycerin and observed with an Olympus fluorescence microscope (Vanox) to search for the localization of prolyl hydroxylase.
Immune reactions were observed with all eight types of antibodies tested, but among them, the strongest was with clone number 3-4H2 (IgM), followed by 3-2B12 (IgG ₁ ). The results for clone number 3-4H2 were observed through photographs, and strong positive findings were observed in fibroblasts in the villous interstitium of the placenta by immunofluorescence. The photograph shows that the immunoreaction was negative in a control specimen using a 1:100 dilution of ascites from a hybridoma that did not produce anti-prolyl hydroxylase antibodies. Example 5 Quantification of prolyl hydroxylase using a combination of single and polyclonal antibodies A polyclonal antibody was prepared by first immunizing white rabbits (female) with human prolyl hydroxylase purified from human placenta in the same manner as in Example 1 together with Freund's complete adjuvant. do. 1 ml of 200 μg of prolyl hydroxylase was subcutaneously administered to 15 points on the back as a mixture with adjuband. For a further 4 months, 200 μg of Freund's complete adjuvant was administered subcutaneously on the back every 2 weeks. After each booster immunization, serum was collected and its antibody titer was measured by the solid phase-antibody binding test described in Example 1(f). In addition,
In this case, the secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG (Miles Laboratories,
Inc.) was used. Anal.Biochem.16, 384−394
When the antiserum obtained according to the method of Hutton et al. (1966) was examined for its inhibitory effect on prolyl hydroxylase activity, 4μ inhibited the activity by 56%. Furthermore, this antiserum was determined to be specific to prolyl hydroxylase since it produced a single sedimentation line in the Ouchterlony immunodiffusion method and the immunoelectrophoresis method. Next, the amount of prolyl hydroxylase in human blood and tissues was measured using the Sanderch method described in the 2nd edition of Enzyme Immunoassay (edited by Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Miyai), Igaku Shoin (1982), 30-49. did. A 96-well vinyl microtitration plate (Falcon 3912) is coated with the previously obtained purified single antibody (2-5G8), and the rest is further blocked with 1.0% BSA. On the other hand, blood and liver tissues of healthy individuals and individuals exhibiting various degrees of liver fibrosis were used as antigen samples. That is, 2 ml of each blood was centrifuged at 200 xg for 5 minutes, and 25 µ of serum as a supernatant was used. In addition, liver tissue was treated with 20 times the amount of 0.25M sucrose per wet tissue weight, and 0.01mM
Contains EDTA and 0.1mM dithiothreitol
It was extracted with 10mM Tris-HCl buffer, pH 7.4, and 5μ of the supernatant after centrifugation (1500×g) was used. These sample solutions are added to antiserum-treated microtitration plates and incubated at room temperature for about 1 hour. Additionally, rabbit antiserum and horseradish peroxidase-conjugated goat anti-rabbit as a secondary antibody were used.
Add IgG and incubate for approximately 1 hour at room temperature. Next, hydrogen peroxide and o-phenylenediamine as a base material were added, and the resulting reddish brown color was quantified using a corona two-wavelength microplate photometer (MTP-22). Figure 4 shows a calibration curve for prolyl hydroxylase, and its detection limit was approximately 2 ng.
In addition, prolyl hydroxylase could be detected in blood and liver tissue by this Sand-Deutsch method, and an increase in the amount of prolyl hydroxylase in the blood associated with human liver fibrosis was observed (FIG. 5). In addition, a single clone with high affinity for the antigen (clone number 3)
-4H2, see Figure 3) It is thought that the Sand Deutsch method using single clones or in combination with other single clones will have a detection sensitivity and accuracy that is equivalent to or even higher than that using polyclonal rabbit antisera (especially 1971-501493). In addition, although enzyme-labeled antibodies were used in this quantification, radioimmunoassay methods or immunological experimental manipulation methods using ^125I -labeled antibodies (single clones and polyclones) were also used.
The enzyme immunoassay method using enzyme-labeled Fab' by Yoshitake et al., described in XI, 3497-3519 (1982), can also be applied to quantify prolyl hydroxylase in a sample. [Table] [Table] [Table]

[Brief explanation of drawings]

Figure 1 shows ascites of clone number 3-2B12 (IgG ₁ ) subjected to ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography treatment, Sephacry1S-
This is a protein elution diagram when gel-filtered with a 300 Superfine column, and Figure 2 shows clone number 2-1C2.
After ammonium sulfate fractionation of ascites (IgG _2b ), ProteinA-
This is a diagram of protein elution during affinity chromatography using a Sepharose CL-4B column, and FIG. 3 is a diagram of the binding of each single antibody purified with human prolyl hydroxylase using a solid phase-antibody binding test method. (――●――)3-2B12, (――▲――)2
−6G9, (――×――)3−4H2, (――○――)2−
5G8, (--▲--)2-1C2, (--×--)3
-3H9, (--○--)3-6H5, (--●--)
2-7F8. FIG. 4 is a calibration curve for human prolyl hydroxylase using a purified single antibody (clone number 2-5G8, IgG ₁ ). In Figure 4,
(――○――) is blocking by 1% BSA,
(――●――) is blocking with 1% obalmine. Ascites fluid from a non-anti-prolyl hydroxylase antibody-producing hybridoma was similarly purified and used as a control. (--○--1% BSA blocking, and --●--1% ovalmin blocking). Figure 5 shows the amount of prolyl hydroxylase in the serum of healthy individuals and patients with various liver diseases.

Claims (1)

[Claims] 1. A monoclonal antibody of the IgG class that has immunocross-reactivity with latoprolyl hydroxylase and has immunocross-reactivity with antigenic determinants present in human prolyl hydroxylase.