Title of Invention	" METHOD FOR QUANTIFYING 1-ACID GLYCOPROTEIN (As AGP) FOR THE PURPOSE OF DIAGNOSING A LIVER DISEASE AND DIAGNOSTIC STRIP FOR IMMUNO CHROMATOGRAPHY"
Abstract	The present invention relates to a method for diagnosing a livar disease rapidly in an early stage. More particularly, the present invention relates to a monoclonal antibody aganist asialo a 1-acid glycoprotin; a method for diagnosing a liver dis- ease which evaluates asialo a I-acid glycopratein in a test sample by using said monoclonal antibody and an diagnostic strip for immunochromatography composed of said monoclonal antibody against XXX a l-acid glycoprotein and Rictons community aggln. tinin ()RCA). The diagnostic devlee of the present invention is convanicnt to measure the concentration of asialo a l-acid glycoprotein and to diagnose a liver disease rapidly.

Title of Invention

" METHOD FOR QUANTIFYING 1-ACID GLYCOPROTEIN (As AGP) FOR THE PURPOSE OF DIAGNOSING A LIVER DISEASE AND DIAGNOSTIC STRIP FOR IMMUNO CHROMATOGRAPHY"

Abstract

The present invention relates to a method for diagnosing a livar disease rapidly in an early stage. More particularly, the present invention relates to a monoclonal antibody aganist asialo a 1-acid glycoprotin; a method for diagnosing a liver dis- ease which evaluates asialo a I-acid glycopratein in a test sample by using said monoclonal antibody and an diagnostic strip for immunochromatography composed of said monoclonal antibody against XXX a l-acid glycoprotein and Rictons community aggln. tinin ()RCA). The diagnostic devlee of the present invention is convanicnt to measure the concentration of asialo a l-acid glycoprotein and to diagnose a liver disease rapidly.

Full Text	MONOCLONAL ANTIBODY AGAINST ASIALO ALPHA 1-ACID GLYCOPROTEIN, IMMUNOCHROMATOGRAFHIC STRIP COMPRISING THE MONOCLONAL ANTIBODY. AND METHOD FOR DIAGNOSING LIVER DISEASES USING THE IMMUNOCHROMATOGRAPHIC STRIP TECHNICAL FIELD The present invention relates to a method for diagnosing a liver disease rapidly in an early stage. More particularly, the present invention relates to a monoclonal antibody against asialo 1-acid glycoprotein, a method for diagnosing a liver disease which, evaluates asialo 1-acid glycoprotein in a test sample by using said monoclonal antibody; and an diagnostic strip for immunochrornatography composed of said monoclonal antibody against asialo 1-acid glycoprotein and Ricinus coumfiunis agglutinirt (RCA) . The diagnostic device of the present invention is convenient to measure the concentration of asialo acid glycoprotein and to diagnose a liver disease rapidly. BACKGROUND Liver disease including hepatitis, liver cirrhosis, and hepatocarcinoma is the most prevalent 1 disease in Korea, Japan, Taiwan, China and other Southeast Asian countries. Presently, liver diseases have been diagnosed by evaluating the content of bilirubin or urobilinogen from patients' urine, by measuring the contents of glutamic-oxaloacetic tranaaminase (GOT), glutamic pyrubic transaminase, total bilirubin, albumin, lactic acid dehydrogenise and the like so as to analyse the changes of biochemical components in blood and by detecting an antigen from hepatitis B virus (HBV) or hepatitis. C viruS (HCV) or antibody against these viruses. Besides, liver cirrhosis can be diagnosed by alpha-feto protein (AFP) and carcinoembryonic antigen (CEA) test. However, liver is a complex organ due to various functions and is vitally specific not to reveal an abnormal state outwardly. Furthermore, an early diagnostic method has not been established yat arid thus livsr disease is often difficult to be treated, since it is diagnosed after severely worsen. The present inventors have developed a marker which diagnoses a liver disease in a early stage' clinically and reflects the severity of patient exactly and then manufactured a diagnostic hit. it bas been disclosed in the patent application and the treatise that the marker be a remarkable agent for diagnosing a liver disease. 2 Precisely, the present inventors have demonstrated the diagnostic method and the diagnostic- kit in Korean patent application PCT/KR00/00840 (Aug. 1, 2000), US patent application 09/662,363 (Sept. 13, 2000) and Korean patent application 10-2000-0040609 (July 14, 3000), which exploits the sandwich ELISA method by usinq the specific antibody ane lectin and measures asialo glycoprotein in blood. This techniques are confirmed to be recurrent and accurate and thus to be useful for diagnosing liver functions and to treat hepatic diseases, It is reported that asialo glycoprotein represent the prognostic status o£ hepatic disease as a marker in blood serum (T. Sawamura et al., Gastroenterology 1981, 81: 527-533; T. Sawamura et al. , Gaetroenterology 1984, 87: 1217-1221) . In addition, it is elucidated that asialo glycoprotein help to detect the status of hepatic cancer since the concentration is proportional to the severity in liver cancer (T. Sawamura et al. , Gsetrologia Japonica 1985, 20: 201-208). Conventionally, the receptor against asialo glycoprotein is separated from human or other animal such as rabbit and mouse, purified and applied as a capture protein in order to measure the concentration of agialo glycoprotein. After it is labeled with radioactive substrates, the competitive radioactive assay and electro immunodiffusion are accomplished (J. 3 S. Marshall et al,, J. Lab. Clin, Med. 1978, 92: 30-37; N. Serbource-Goguel et al., Hepatology 1933, 3: 356-359) . Unfortunately, there are some problems. Above all, the receptor against asialo glycoprotein is difficult to be obtained in a large scale, although the test kit needs a large amount of asialo glycoprotein. In case of competitive radioreceptor assay, it is dangerous to use radioactive substance and hard to prepare special facilities for treating waste material and the like. In cases of electroimmunodiffusion, it is complicated to analyst data quantitatively. Especially, the competitive assay is not suitable for general diagnostic kit since it lacks accuracy and recurrence. BHIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and other advantages of the prsssnt invention will bs wire clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which; FIG. 1 depicts 1-acid glycoprotein (AGP) and desialylated 1-acid glycoprotein purified from blood plasma by performing sodium dmiecyl polyacrylamide gel electrophoresis (SDS-PAGE). 4 FIG. 2 depicts the monoclonal antibody against asialo 1-acrid glycoprotein produced from the hybridoma cell line of the present invention by performing; western blotting. FIG. 3 depicts the monoclonal antibody prepared in the present invention by performing ELISA method, which reacts only with asialo 1-acid glycoptotein and excludes heptoglobin and 2-macroglobulin. FIG 4a depicts a planar view of the diagnostic strip for immunochromatography prepared in the present invention. FIG 4b depicts a front view of the diagnostic strip for immunochromatography prepared in the present invention. FIG 5 depicta the result which is measured by using the cassette type diagnostic strip for immunochromatography prepared in the present invention and indicates asialo 1-acid glycoprotein in a. concetration- dependent mode. DISCLOSURE OF THE INVENTION 5 In order to settle above-mentioned technical problems and to diagnose a livsr disease rapidly and easily by detecting asialo gly coprotein, the present inventors have attempted to develop a monoclonal antibody specific for asialo 1-acid glycoprotein (AsAGP), a diagnostic method for liver disease by using said monoclonal antibody and a diagnostic strip for immunochromatography useful for the same method. The object of the present invention is to provide a device and a method for detecting a livsr disease easily. In order to attain said object, the present invention provides a monoclonal antibody binding only with asialo 1-acid glycoprotein and excluding heptoglobin and 1-macroglobulin. The monoclonal antibody of the present invention also does not react asialo heptcglobin and asialo 1-macroglobulin. Preferably, the monoclonal antibody of the present invention is a subclass type IgG1, The monoclonal antibody can be prepared by the process disclosed in the prior arts (Davidson R. L. and P. S. Gerald 1976, Improved techniques for the induction. of mammalian cell hybridisation by polyethylene glycol, Somatic Cell Genet., 2: 165-176; Knott C. L., Kuus-Reichel K. , Liu R. and Wolfert R.L. 1997, Development of antibodies for diagnostic assays, In Price C. and Newman D. (eds.) Principles and 6 Practice of Immunoassay, 2nd ed. New York, Stockton Press, 36-64; Gillete R. W. 1987, Alternatives to pristine priming for ascitic fluid and monoclonal antibody production, J. Immunol. Meth. 39, 21-23, Morwood T. H. , C. J. Zeigler and G. M. Martin 1976, Dimethyl sulphoxide enhances polyethylene glycol- mediated somatic cell fusion, Somatic cell Genet., 2: 263-270) Precisely, it is manufactured by the process which comprises (1) separating asialo 1-acid glycoprotein and (2) immunising mice. Above all, in order to produce the monoclonal antibody obtained above in a large scale, the hybridoma cell is prepared by the conventional process, separated, screened, injected into a mouse peritoneally and then collected from peritoneal fluid. Concretely, asialo 1-acid glycoprotein is purified from blood by the process as described in the patent applications [PCT application PCT/KR00/00840 (Aug, 1, 2000), US patent application 09/662,363 (Sept, 13, 2000), Korean patent application 10-3000-0040609 (July 14, 2000)]. Asialo i-acid glycoptotein is auapended by using phosphate buffer, blended by using Titer-MAX and applied to immunize a mouse. A spleen cell and a myeloma cell are separated from the experiniental mouse immunized, fused and screened to select a hybridoina cell line specific for asialo 1- acid glycoprotein by BLISA method. In order to produce 7 the monoclonal antibody specific for asialo 1-acid glycoprotein in a large scale from the hybridoma cell above, the hybridoma cell producing the monoclonal antibody against asialo 1-acid glycoprotein is injected to experimental mice and the peritoneal fluid of the mice containing the hybridama cell in a high level is collected and separated a cell specific for the monoclonal antibody of the present invention. In addition, the present invention also provides the hybridoma cell line which can produce in a large seals a monoclonal antibody binding only with asialo 1-acid glycoprotein excluding heptoglobin and macroglobulin. In order to investigate whether the monoclonal antibody against asialo 1-acid glycoprotein obtained from the hybridoma cell be specific for asialo 1-acid glycoprotein or not, asialo 1-acid glycoprotein is first analyzed by performing electrophoresie and western blotting, it is further examined whether the monoclonal antibody of the present invention reacts only with asialo 1-acid glycoprotein and excludes other glycoproteing by performing ELISA method and the like as demonstrated in Example 2, Preferably, the present invention provides the hybridoma cell line, producing the subclass type IgGI monoclonal antibody specific for asialo 1-acid glycoprotein and deposited to Korea Research Institute 8 of Bioscience and Biotechnology, Gene Bank in May 24, 2004 (accession number KCTC 10251 BP) under Budapest Treaty. In addition, the present invention provides a. method for detecting a hepatic disease which comprises steps (l) react ing a monaclonal antibody which binds only with asialo 1-acid glycoprotein and excludes heptoglobin and 2-macroglobulin; Ricinus comntunis agglutinin (hereinafter, referred to as "RCA") as a lectin, specifically recognising asialo glycoprotein; and a test sample; and (2) measuring asialo 1-acid glycoprotein (AsAGP). In the method for detecting a hepatic disease of the present invention, test samples can be analysed on a microplate through a sandwich enzyme immunoassay, an enzyme immunoassay onto an diagnostic strip for immunochromatography and various types of enzyme immunoassay. Especially, the enzyme immunoassay onto diagnostic strip fur immunochromatography is the most convenient among these methods. Preferably, the monoclonal antibody utilized in the diagnostic method of the present invention is a subclass type IgGI as described above. More preferably, the monoclonal antibody is produced from the mouse hybridoma cell line of the present invention deposited with accession number KCTC 10261 BP. Besides, as a lectic, RCA recognising asialo 1-acid glycoprotein is 9 preferable to be utilized. The present invention provides a diagnostic scrip for immunochromatography which comprises a monoclonal antibody binding only with asialo 1-acid glycoprotein and excluding heptoglobin and 2-macroglobulin; and lectin RCA recognizing asialo glycoprotein; measure the concentration of asialo 1-acid glycoproteir. in a test sample; and diagnose a liver disease rapidly and easily. Preferably, the present invention provides the diagnostic strip for immunochromatography which includes the monoclonal antibody As 16.85 deposited with the accession number KCTC 102 61 BP and RCA as a lectin. The diagnostic strip for immunchromatography of the present invention comprises glass fiber (GF) tnerobrane coated with micro-particles such as gold- colloid conjugated with monoclonal antibody; nitrocellulose membrane (NC) in which RCA band is lined as a diagnostic line and an monoclonal antibody band as a standard line; a sample pad absorbing test sample solution; an absorbent pad discarding non-reactive substance; and an adhesive plastic backing for mounting the above-mentioned members. The diagnostic strip for immunochroniasography of the present invention is mounted in due turn, preferably NC membrane, GF membrane, a sample pad and absorbent pad are partially overlaid onto the adhesive 10 plastic backing to transfer substance smoothly by capillary reaction. The diagnostic strip for immunochromatography can be prepared by the conventional procedure. Preferably, the diagnostic strip of the present invention can be manufactured to a cassette type or a stick type. Besides, the test sample is preferable to be blood or serum and 10-fold diluted by using elution buffer and the elution buffer is preferable to be 50 mM borate buffer containing 5% sucrose, 1% bovine serum albumin or 1% Triton X-100. The diagnostic strip for immunocrhromatography of the present invention is used to detect a liver disease as follows: when a test sample is diluted and dropped onto the sample pad, both the standard line and diagnostic line are colored on the atrip after 3-5 minutes. SO that the sample be judged according to colors whether positive for hepatic disease or not. Precisely, if Ab-gold conjugate is adopted as a microparticle, the positive sample including asialo 1- acid glycoprotein reveals red color both on the standard line and the criteria line after a test and the negative sample including asialo 1-acid glyeoprotein in a normal level (not a patient of liver disease) reveals red color only on the standard line. As described above, the diagnostic strip for immunochromatography of the present invention can 11 detect asialo 1-acid glycoprotein to reach a cutoff value, sbout 1.50 g/ml, which can diagnose a liver disease early and monitor its prognosis and treatment if the level of asialo 1-acid glycoprotein present in patients of liver cirrhosis and liver cancer continued to be evaluated, Furthermore as illustrated above, the monoclonal antibody which reacts only with asialo 1-acid glyeoprotein and excludes heptoglobin and 2- macroglobulin and the diagnostic atrip for immunochromatagraphy by using the same can inform the test result rapidly and conveniently so that liver diseases can be diagnosed easily. Therefore, it is expected to contribute to the prevention and treatment of liver diseases EXAMPLES Practical and presently preferred embodiments of the present invention are illustrated as shown in the following Examples. However, it will be appreciated chat those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention. Separation and purification of asialo 1- 12 acid glycoprotein (AaAGP) 1-acid glycoprotein (AGP) was separated and purified from asialo glycaprotein contained in human blood plasma as follows. 2 NIH unit of thronbin was added to 20 0 ml of human blood plasma, stored at 37 C for 2 hours and at 4C overnight and centrifuged to remove blood clot. The serum prepared above was dialyzed by using 0,05M of sodium acetate buffer (pH 4.3), loaded onto DEAE column previously equilibrated by using the same buffer, and then eluted through linear concentration gradient after 0.05 M of sodium acetate buffer (pH 4.3) and 0.1 M of sodium acetate buffer (pH 4.3) were mixed. Afterward, the optical density (OD) was measured at 280 nm and the data was illustrated in FIG. 1. The fractions including AGP and other proteins were collected, mixed with antmonium stilfate to 0.5 g/ml and centrifuged to precipitate proteins. The resulting supernatant was mixed again with ammonium sulfate bo 0.18 g/ml to precipitate proteins and the pellet was dissolved in a small amount of distilled water (D, W.), dialysed sufficiently by using D. W. and then lyophilieed. 40 mg of AGP separated above was hydrolyzed at 80C for 2 hours by using 6 ml of 0.1 N sulfate solution, neutralized by using 1 N of sodium hydroxide and then dialysed by using 0,01 N of phosphate buffer 13 (pH 7,4) . The desialylaced 1-acid glycoprotein (AsAGP) prepared in the above procedure was loaded onto Sephadex G-200 column, filtrated by using 0.01 M of phosphate buffer (pH 7.4) and calculated through OD value at 280 nm and finally the fractions including proteins were collected. The result was illustrated in FIG. 1: lane 1 is the standard marker of protein molecular weight; lane 2 is 1-acid glycoprotein; lane 3 and 4 are asialo 1-acid glycaprotein (AsACP). Preparation of monoclonal antibody against asialo 1-acid glycoprotein (1) Immunization of mice In order to obtain an, immunised mouse essential to prepare a hybridoma cell line producing a monoclonal antibody against asialo 1-acid glycoprotein, asialo 1-acid glycoprotein as an antigen was suspended well by using Titer-MAX, adjusted to 50 g/50 ml of concentration and then injected into peritoneal cavity of Balb/c mice aged 6-8 weeks. After 2 weeks, the same amount o£ antigen mentioned in the first injection was mixed with Titer-MAX and injected repeatedly onto the same site. Through the same procedure, the antigen was injected again after 7 days and repeatedly injected after 3 weeks. Afterward, the small amount of blood was collected from a tail of mouse and examined to evaluate 14 a titer. (2) Cell fusion In order to perform cell fusion for preparing a hybridoma cell line, mice were immunised with asialo 1-acid glycoprotein antigen mentioned above and spleen cells and myeloma cells were collected front the mice. Then, 103 of spleen cell and 107 myeloma cell (SP2/0) were washed sufficiently, mixed together and 1 ml of PEG 1500 was poured for about 1 minutes, stirred slightly for about 1 minutes. Afterward, 9 ml of RPMI medium was added for about 3 minutes to reach 50 ml of final volume as stirred slowly. The cell suspension was centrifuged to collect the cell pellet, suspended Again to 1-2 X 103 cells/ml by using HAT medium, poured into a 96-well microplate in 0.2 ml volume per well, and then incubated at 37 C in CO2 incubator, (3) Screening of hybridoma cell producing monoclonal antibody In order to select a hybridoma cell specific for asialo 1-acid glycoprotein, ELISA method was tried by exploitrig a microplate coated with asialo 1-acid glycoprotein as follows. Precisely, asinlo 1-acid glycoprotein antigen was put into a microplate in 100 1 (1 g/ml) per well to coat the surface and washed off to remove antigens not reacted. The culture medium 15 of hybridoma cell was poured to each well in 100 1, reacted for 2 hours and washed off by using Tween 20 phosphate buffer (PBST) Co remove the culture medium not reacted. Afterward, goat anti-mouse IgG-horseradish peroxidase (HRP) was added, reacted for an hour at room temperature and washed off by using PBST solution. Then, ortho-phenylenediamine (OPD) was utilized, as a substrate of peroxidase, reacted and examined to measure OD value at 490 nm with ELISA reader. As a result, the cell line secreting antibodies highly binding to asialo 1-acid glycoprotein antigen was first screened and repeatedly selected to separate the hybridoma cell secreting a monoclonal antibody specific for asialo 1-acid glycoprotein antigen. The resulting cells were treated by the limiting dilution to become a monoclone and the hybridoma, cell line producing a monoclonal antibody was sorted again. Afterward, 2 clones having the highest titer such as asialo 1-acid glycoprotein 1 and asialo 1-acid glycoprotein 2 were collected, tried to measure the titer of supernatant in culture medium through ELISA method and then the subunit type of the monoclonal antibody Was analyzed by the double immunodiffusion, Consequently, the antibodies secreted from the clones. of the present invention were identified as IgGI and IgM reapectively. The cell line secreting IgGI has been named aa AS 16 16.89 said deposited to Korea Research Institute of Bioecience and Biotechnology, Gene Bank in May 24, 2004 (accession number KCTC 10261 BP) . (4) Large scale production and purification of monoclonal antibody In order to produce a monoclonal antibody from a hybridoma cell in a large scale, 0.5 ml of pristane was injected into peritoneal cavity of Balb/c mice. After 1 week, each cell line obtained in the above procedure (3) was injected to the experimental mice in 5 x l06 cells per mouse and then peritoneal fluid was collected from the peritoneal cavity if swollen. The peritoneal fluid was centrifuged at 12,000 rpm to collect cells and the resulting supernatant was filtrated through protein G column to separate and purify the monoclonal antibody against asialo 1-acid glycoprotein and, also stored at -20 C for the next experiments. (5) Identification of specificity of monoclonal antibody by western blot In order to elucidate the property of monoclonal antibody specific for asialo 1-acid glycoprotein, the monoclonal antibody against asialo 1-acid glycoprotein purified through the above-mentioned procedure (4) was identified by performing SDS- polyacrylamide gel electrophoresis and western blotting 17 [See FIG 2) . The western, blotting was accomplished by a typical procedure in the prior arts, in FIG. 2, lane 1 is a standard marker of protein molecular weight; lane 2, 1-acid glycoprotein; Lane 3, asialo 1-acid glycoprotein; lane 4, 2-macroglobulin; and lane 5, heptoglobin. (6) Identification of specificity of monoclonal antibody by enzyme immunochemical assay Asialo 1-acid glycoprotein, heptoglobin, 2- macroglobulin antigen were added to 96-well microplate in 100 1 (1 g/ml) per well respectively, attached on the surface, reacted for 2 hours with the monoclonal antibody against asialo 1-acid glycoprotein as added in 100 1, and washed off by using phosphate buffer- Tween 20 (PBST) to remove the culture medium not reactfed. Further, goat anti-mouse IgG-HRP was added, reacted for an hour at room temperature and washed sufficiently by using PBST solution. Afterward, ortho- phenylenediamine (OPD) was added as a substrate of peroxidase and reacted so as to measure OD value at 490 nm with ELISA reader. As a result, it is confirmed that the monoclonal antibody against asialo 1-acid glycoprotein purified above be the monoclonal antibody specific for asialo 1-acid glycoprotein. {See FIG. 3} . In FIG. 3, AGP depicts 1-acid glycoprotein and AsAGF, asialo 1-acid glycoprotein of the present invention. 18 Detection of liver disease by diagnostic kit for immunochromatography The diagnostic kit for immunochromatography of the present invention which can measure the concentration of asialo 1-acid. glycoprotein in a test sample by the immune reaction between, the monoclonal antibody. As 16.89 specific far asialo 1-acid glycoprotein produced above and asidlo 1-acid glycoprotein in blood was manufactured and applied in clinical fields as follows. (1) Preparation of diagnostic kit for immunochromatography The diagnostic kit for detecting asialo 1-acid glycoprotein in blood is composed of following components as described below. A. monoclonal antibody (As16.89) specific for asialo 1-acid glycaprotein fixed onto solid carrier such as microplate 19 B. lectin. (RCA) -horseradish peroxidase (HBP) against asialo 1-acid glycoprotein C. sample dilution buffer (1% BSA/PEST) D. enzyme eubstrate solution (OPD) E. washing buffer (PBST) F. standard solution of asialo 1-acid glycoprotein G. stopping buffer (2) Meagurement a. The standard solution (1 g/ml) of asialo 1- acid glycoprotein was poured in 100 1 respectively to mieroplate on which the monoclonal antibody As 16.89 was fixed. Experiment samples, including 40 normal persons, 155 patients without liver disease, 36 patients of acute hepatitis, 272 patients of chronic hepatitis (CH), 230 patients of liver cirrhosis, 72 patients of hepatocarcinoma (HCC) proceeded from liver 20 cirrhosis, were obtained from St. Mary Hospital, Catholic Medical School. The blood sample was diluted to 1 : 10 ratio by using the dilution buffer, allotted onto each well of microplate in 100 1 and incubated for 120 minutes at room temperature. b. After reacted, the washing buffer was added in 100 1 per well and washed three times repeatedly. c. The lectin (RCA)-HRP diluted by the process for the preparation was allotted onto each well of microplate and reacted for 60 minutes at room temperature. d. Step b was repeated. e. The enzyme substrate solution (OPD) was allotted onto each well of microplate above. f. The stopping buffer was added on each well in 10 0 1 to stop the ensymatic reaction. g. OD value was measured at 490 nm with RLISA reader. As a result, the content of asialo 1-acid glycoprotein in blood was evaluated through the above- 21 mentioned procedure as illustrated in Table 1. 22 chronic hepatitis, liver cirrhosis and hepatocarcinema, with liver cirrhosis, the blood level of AsAGP was evaluated to average 1.33 g/ml, 1.63 g/ml, 3.12 g/ml and 3.64 g/ml respectively. The concentrations be remarkably distinguished from those of standard group, even if the ratio of sample over 1.5 g/ml of AsAGP be 25%, 36%, 72% and 82% respectively, Therefore, it is verified that this test be effective upon the diagnosis of hepatic diseases. Hence, according to data demonstrated above, the cutoff value for diagnosing a liver disease was decided to be 1.50 g/ml when the monoclonal antibody As 16.39 of the present invention was used for the enzyme immunoassay. Preparation of diagnostic strip for immunochromatography. (1) Preparation of monoclonal antibody-gold conjugate The monoclonal antibody specific for asialo 1- acid glycoprotein selected in the present invention was added in 15 g/ml to the colloidal solution of gold particles, reacted for 2 hours at room tempsrature and rotated- Then, 10% of BSA was blended in 1/10 volume to become 1% of concentration and again reacted for an hour to prepare Ab-gold conjugate. Afterward, the 25 resultant was centrifuged at 12,000 rpm for 40 minutes to discard the supernatant solution and 2 mM of borate buffer was added to wash off Ab-gold conjugate. The resultant wag washed repeatedly, three times and finally 2 mM of borate buffer containing 1% BSA was added in about 1/10 volume of gold solution and suspended. After measured with UV spectrometer, the OD value at 530 nm was adjusted to 3.00 by dilution. (2) Sample pad The sample pad is a member absorbing test sample and is made of cellulose materia1 for this purpose. (3) Glass fiber (GF) metobrane The glass fiber is a component on which AsAGP in tesc sample and the monoclonal antibody prepared by the process of the present invention are reacted. The GF membrane was coated with gold colloid particle-As 16.89 on the surface and prepared as follows. The monoclonal antibody produced from hybridoma call line. As 16.39 of the present invention was conjugated onto the membrane as illustrated in step (1). GF membrane (1.0 cm X 0.7 cm) purchased from Millipore Co. Ltd, was soaked by using 20 mM sodium borate buffer, sprayed uniformly by using gold colloid particle-As 16.89 onto the surface, dried at 37 C and 26 thus made to GF membrane pad for immunochromatography (also conjugate pad) on which a monoclonal antibody conjugated with a coloring particle is fixed. (4) Nitrocellulose (NC) membrane and lining Nitrocellulose membrane purchased from Millipore Co. Ltd. was cut to a proper size (0.7 cm X 5 cm) , lined at a point of about 2.4 cm from the bottom of plaetic backing with goat anti-mouse IgG as a standard line and at a point of 2.7 cm as a criteria line for detecting asialo 1-acid glycoprotein by using RCA purchased from EY Lab, and then dried to complete the NC membrane. (5) Absorbent pad Cellulose membrane was used to absorb substance not reacted in a test sample after an immune reaction. and to transfer the sample solution through capillary reaction. (6) Preparation of diagnostic strip for immunchromatography. As illustrated in FIG. 4a and FIG. 4b, the above- mentioned members were mounted onto the adhesive plastic backing. Precisely, NC membrane, GF membrane, sample pad and absorbent pad were arranged in due turn, overlaid evenly in about 0.1 cm portion and fixed by 27 adhesion in order to transfer substance smoothly through capillary reaction, Analysis of asialo 1-acid glycsprotein by using diagnostic strip for immunochromatography Sarum sample was diluted to 1 : 10 ratio by using elution buffer (such as 50 mM borate buffer containing 5% sucrose, 1% bovine serum albumin or 1% Triton X-100) and added in 60 - 70 I volume onto the sample pad of diagnostic atrip for immunochramatography prepared in Example 4 so as to examine the standard line and the criteria line after 3 - 5 minutes whether colored or not. FIG. 5 depicts the result from the cassette type strip for immunochramatography. NO. 1 is the standard dropping the mixture of 1-acid glycoprotein (AGP), heptoglobin and 2-macraglobulin; NO. 2, the diagnostic strip in the serum of normal person; NO. 3 -NO- 6, the strip id 1.5 g/ml, 2.0 g/ml, 3.0 g/ml and 4.0 g/ml of asialo 1-acid glycoprotein respectively. As illustrated in FIG. 5, in NO. 3, the line is obscure; and in MO. 4 - NO- 6, red colors are clear both on the standard line (upper) and the criteria line (lower). The diagnostic strip for immunochramatography of the present invention is observed that only the standard line become red in the test sample of normal 28 person (negative) under the cutoff value, 1.50 g/ml of asialo 1-acid glycoprotein since antigen-antibody- enzyme conjugate complex wag not formed. On the other hand, when asialo 1-acid glycoprotein level increased bo more than 1.50 g/ml due to a liver disease, both the lines become red, in which asialo 1-acid glycoprotein was absorbed on the sample pad, reacted with gold particle-monoclonal antibody As 16.89 conjugate on GF membrane to form an immune complex, transferred onto NC membrane through, capillary reaction and conjugated with RCA in the criteria line on NC membrane to make gold precipitate. Furthermore, the criteria line can be varied in thickness and color density, depending upon the concentration of asialo 1- acid glycoprotein. Therefore, it is possible to predict the severity of liver disease by the concentration of asialo 1-acid glycoprotein in the test sample. INDUSTRIAL APPLICABILITY As illustrated above, the monoclonal antibody specific for asialo 1-acid glycoprotein and the diagnostic strip and the diagnostic kit for immunochramatography by using the same of the present invention can be utilized to evaluate asialo 1-acid glycoprotein present in blood sample rapidly and easily. Therefore, the monoclonal antibody and the 29 diagnostic device of the present invention can monitor the severity, the treatment and Che prognosis of liver diseases efficiently. Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims. 30 What is claimed is: 1. A monoclonal antibody which reacts only with asialo 1-acid glycoprotein. and excludes heptoglobin and 2-macroglobulin. 2. The monoclonal antibody according to claim 1, which is a subclass type IgGI. 3. A cell line which produces in a large scale a monoclonal antibody reacting only with asialo 1-acid glycoprotein and excluding heptoglobin and 2- macroglobulin. 4. The cell line according to claim 3, which is prepared by fusing a spleen cell and a myeloma cell extracted from a mouse immunised with asialo 1-acid glycoprotein. 5. The cell line according to claim 4, which produces the subclass type IgGI monoclonal antibody specific for asialo 1-acid glycoprotein and is deposited to Korea Research Institute of Bioscience and Biotechnology, Gane Bank in May 24, 2004 (accession number KCTC 10349 BP) under Budapest Treaty. 6. A method for diagnosing a liver disease in which 31 a monoclonal antibody which reacts only with asialo 1- acid glycoprotein and excludes heptcglotin and 1- macroglobulin; and lectin RCA (Ricinua communis agglutinin) recognizing asialo glycoprotein are reacted with a test sample to measure the amount of asialo 1- acid, glycoprotein (AsAGP). 7. The method for diagnosing a liver disease according to claim 6, in which the monoclonal antibody is the subclass IgGI produced from the mouse cell line deposited with the accession number KCTC 10261 BP. 8. The method for diagnosing a liver disease according to claim 6, in which the test sample is blood or serum. 9. An diagnostic strip for immunochramatography which comprises a monoclonal antibody reacting only with asialo 1-acid glycoprotein excluding heptoglobin and 2-macroglobulin; and RCA recognizing asialo glycoprotein as a lectin; and reacts a test sample at over 1.50 g/ml of asialo 1-acid glycoprotein to detect a liver disease. 10. The diagnostic strip for immunochromatography according to claim 9, in which the monoclonal Antibody is As 16.89 deposited with the accession number KCTC 32 10261 BP. 11. The diagnostic strip for immunochramatography according to claim 9, in which the monoclonal antibody- is conjugated with a micro-particle such as colloid type gold particle. 12. The diagnostic strip for immunochramatography according to claim 11, which comprises (1) glass fiber- (GF) coated with, micro-particles conjugated with the monoclonal antibodys (2) nitrocellulose membrane (NC) including a standard line and a diagnostic line to detect a glycoprotein; (3) a sample pad for a test sample; (4) a absorbent pad for non-reactive substance; and (4) an adhesive plastic backing for imountiag above- mentioned naembers. 13. The diagnostic strip for immunochromatography according to claim 12, in which NC meaibrane, GF membrane, a sample pad and a absorbent pad are partially overlaid on said adhesive plastic backing in due turn to transfer substance by capillary reaction and an RCA band as a diagnostic line and an antibody band as a standard line are separated in some interval. 14. The diagnostic- strip for immunochramatography according to claim 9, in which said beet sample is 33 20. The diagnostic kit for immunochramatography according to claim 19, in which, the monoclonal antibody is As 16.89 deposited with the accession number KCTC 10261 BP. 35 The present invention relates 10 a method for diagnosing; a livar disease rapidly in an early stage. More particularly, the present invention relates to a monoclonal antibody aganist asialo a 1-acid glycopreting; a method for diagnosing a liver dis- ease which XXX asialo a I-acid flycopratein in a test sample by asing said monoclonal antibody; and an diagnostic strip for XXX composed of said monoclonal antibody against XXX a l-acid glycoprotein and Rictons community aggln. tinin ()RCA). The diagnostic devlee of the present invention is convanicnt to measure the concentration of asialo a l-acid glycoprotein and to diagnose a liver disease rapidly.

Full Text

MONOCLONAL ANTIBODY AGAINST ASIALO ALPHA 1-ACID
GLYCOPROTEIN, IMMUNOCHROMATOGRAFHIC STRIP
COMPRISING THE MONOCLONAL ANTIBODY. AND METHOD
FOR DIAGNOSING LIVER DISEASES USING THE
IMMUNOCHROMATOGRAPHIC STRIP
TECHNICAL FIELD
The present invention relates to a method for
diagnosing a liver disease rapidly in an early stage.
More particularly, the present invention relates to a
monoclonal antibody against asialo 1-acid
glycoprotein, a method for diagnosing a liver disease
which, evaluates asialo 1-acid glycoprotein in a test
sample by using said monoclonal antibody; and an
diagnostic strip for immunochrornatography composed of
said monoclonal antibody against asialo 1-acid
glycoprotein and Ricinus coumfiunis agglutinirt (RCA) . The
diagnostic device of the present invention is
convenient to measure the concentration of asialo
acid glycoprotein and to diagnose a liver disease
rapidly.
BACKGROUND
Liver disease including hepatitis, liver
cirrhosis, and hepatocarcinoma is the most prevalent
1

disease in Korea, Japan, Taiwan, China and other
Southeast Asian countries. Presently, liver diseases
have been diagnosed by evaluating the content of
bilirubin or urobilinogen from patients' urine, by
measuring the contents of glutamic-oxaloacetic
tranaaminase (GOT), glutamic pyrubic transaminase,
total bilirubin, albumin, lactic acid dehydrogenise and
the like so as to analyse the changes of biochemical
components in blood and by detecting an antigen from
hepatitis B virus (HBV) or hepatitis. C viruS (HCV) or
antibody against these viruses. Besides, liver
cirrhosis can be diagnosed by alpha-feto protein (AFP)
and carcinoembryonic antigen (CEA) test. However, liver
is a complex organ due to various functions and is
vitally specific not to reveal an abnormal state
outwardly. Furthermore, an early diagnostic method has
not been established yat arid thus livsr disease is
often difficult to be treated, since it is diagnosed
after severely worsen.
The present inventors have developed a marker
which diagnoses a liver disease in a early stage'
clinically and reflects the severity of patient exactly
and then manufactured a diagnostic hit. it bas been
disclosed in the patent application and the treatise
that the marker be a remarkable agent for diagnosing a
liver disease.
2

Precisely, the present inventors have
demonstrated the diagnostic method and the diagnostic-
kit in Korean patent application PCT/KR00/00840 (Aug.
1, 2000), US patent application 09/662,363 (Sept. 13,
2000) and Korean patent application 10-2000-0040609
(July 14, 3000), which exploits the sandwich ELISA
method by usinq the specific antibody ane lectin and
measures asialo glycoprotein in blood. This techniques
are confirmed to be recurrent and accurate and thus to
be useful for diagnosing liver functions and to treat
hepatic diseases,
It is reported that asialo glycoprotein represent
the prognostic status o£ hepatic disease as a marker in
blood serum (T. Sawamura et al., Gastroenterology 1981,
81: 527-533; T. Sawamura et al. , Gaetroenterology 1984,
87: 1217-1221) . In addition, it is elucidated that
asialo glycoprotein help to detect the status of
hepatic cancer since the concentration is proportional
to the severity in liver cancer (T. Sawamura et al. ,
Gsetrologia Japonica 1985, 20: 201-208).
Conventionally, the receptor against asialo
glycoprotein is separated from human or other animal
such as rabbit and mouse, purified and applied as a
capture protein in order to measure the concentration
of agialo glycoprotein. After it is labeled with
radioactive substrates, the competitive radioactive
assay and electro immunodiffusion are accomplished (J.
3

S. Marshall et al,, J. Lab. Clin, Med. 1978, 92: 30-37;
N. Serbource-Goguel et al., Hepatology 1933, 3:
356-359) .
Unfortunately, there are some problems. Above
all, the receptor against asialo glycoprotein is
difficult to be obtained in a large scale, although the
test kit needs a large amount of asialo glycoprotein.
In case of competitive radioreceptor assay, it is
dangerous to use radioactive substance and hard to
prepare special facilities for treating waste material
and the like. In cases of electroimmunodiffusion, it is
complicated to analyst data quantitatively. Especially,
the competitive assay is not suitable for general
diagnostic kit since it lacks accuracy and recurrence.
BHIEF DESCRIPTION OF THE DRAWINGS
The above and other objects, features and other
advantages of the prsssnt invention will bs wire
clearly understood from the following detailed
description taken in conjunction with the accompanying
drawings, in which;
FIG. 1 depicts 1-acid glycoprotein (AGP) and
desialylated 1-acid glycoprotein purified from blood
plasma by performing sodium dmiecyl polyacrylamide gel
electrophoresis (SDS-PAGE).
4

FIG. 2 depicts the monoclonal antibody against
asialo 1-acrid glycoprotein produced from the hybridoma
cell line of the present invention by performing;
western blotting.
FIG. 3 depicts the monoclonal antibody prepared
in the present invention by performing ELISA method,
which reacts only with asialo 1-acid glycoptotein and
excludes heptoglobin and 2-macroglobulin.
FIG 4a depicts a planar view of the diagnostic
strip for immunochromatography prepared in the present
invention.
FIG 4b depicts a front view of the diagnostic
strip for immunochromatography prepared in the present
invention.
FIG 5 depicta the result which is measured by
using the cassette type diagnostic strip for
immunochromatography prepared in the present invention
and indicates asialo 1-acid glycoprotein in a.
concetration- dependent mode.
DISCLOSURE OF THE INVENTION
5

In order to settle above-mentioned technical
problems and to diagnose a livsr disease rapidly and
easily by detecting asialo gly coprotein, the present
inventors have attempted to develop a monoclonal
antibody specific for asialo 1-acid glycoprotein
(AsAGP), a diagnostic method for liver disease by using
said monoclonal antibody and a diagnostic strip for
immunochromatography useful for the same method.
The object of the present invention is to
provide a device and a method for detecting a livsr
disease easily.
In order to attain said object, the present
invention provides a monoclonal antibody binding only
with asialo 1-acid glycoprotein and excluding
heptoglobin and 1-macroglobulin. The monoclonal
antibody of the present invention also does not react
asialo heptcglobin and asialo 1-macroglobulin.
Preferably, the monoclonal antibody of the present
invention is a subclass type IgG1,
The monoclonal antibody can be prepared by the
process disclosed in the prior arts (Davidson R. L. and
P. S. Gerald 1976, Improved techniques for the
induction. of mammalian cell hybridisation by
polyethylene glycol, Somatic Cell Genet., 2: 165-176;
Knott C. L., Kuus-Reichel K. , Liu R. and Wolfert R.L.
1997, Development of antibodies for diagnostic assays,
In Price C. and Newman D. (eds.) Principles and
6

Practice of Immunoassay, 2nd ed. New York, Stockton
Press, 36-64; Gillete R. W. 1987, Alternatives to
pristine priming for ascitic fluid and monoclonal
antibody production, J. Immunol. Meth. 39, 21-23,
Morwood T. H. , C. J. Zeigler and G. M. Martin 1976,
Dimethyl sulphoxide enhances polyethylene glycol-
mediated somatic cell fusion, Somatic cell Genet., 2:
263-270) Precisely, it is manufactured by the process
which comprises (1) separating asialo 1-acid
glycoprotein and (2) immunising mice.
Above all, in order to produce the monoclonal
antibody obtained above in a large scale, the hybridoma
cell is prepared by the conventional process,
separated, screened, injected into a mouse peritoneally
and then collected from peritoneal fluid.
Concretely, asialo 1-acid glycoprotein is
purified from blood by the process as described in the
patent applications [PCT application PCT/KR00/00840
(Aug, 1, 2000), US patent application 09/662,363 (Sept,
13, 2000), Korean patent application 10-3000-0040609
(July 14, 2000)]. Asialo i-acid glycoptotein is
auapended by using phosphate buffer, blended by using
Titer-MAX and applied to immunize a mouse. A spleen
cell and a myeloma cell are separated from the
experiniental mouse immunized, fused and screened to
select a hybridoina cell line specific for asialo 1-
acid glycoprotein by BLISA method. In order to produce
7

the monoclonal antibody specific for asialo 1-acid
glycoprotein in a large scale from the hybridoma cell
above, the hybridoma cell producing the monoclonal
antibody against asialo 1-acid glycoprotein is
injected to experimental mice and the peritoneal fluid
of the mice containing the hybridama cell in a high
level is collected and separated a cell specific for
the monoclonal antibody of the present invention.
In addition, the present invention also provides
the hybridoma cell line which can produce in a large
seals a monoclonal antibody binding only with asialo
1-acid glycoprotein excluding heptoglobin and
macroglobulin.
In order to investigate whether the monoclonal
antibody against asialo 1-acid glycoprotein obtained
from the hybridoma cell be specific for asialo 1-acid
glycoprotein or not, asialo 1-acid glycoprotein is
first analyzed by performing electrophoresie and
western blotting, it is further examined whether the
monoclonal antibody of the present invention reacts
only with asialo 1-acid glycoprotein and excludes
other glycoproteing by performing ELISA method and the
like as demonstrated in Example 2,
Preferably, the present invention provides the
hybridoma cell line, producing the subclass type IgGI
monoclonal antibody specific for asialo 1-acid
glycoprotein and deposited to Korea Research Institute
8

of Bioscience and Biotechnology, Gene Bank in May 24,
2004 (accession number KCTC 10251 BP) under Budapest
Treaty.
In addition, the present invention provides a.
method for detecting a hepatic disease which comprises
steps (l) react ing a monaclonal antibody which binds
only with asialo 1-acid glycoprotein and excludes
heptoglobin and 2-macroglobulin; Ricinus comntunis
agglutinin (hereinafter, referred to as "RCA") as a
lectin, specifically recognising asialo glycoprotein;
and a test sample; and (2) measuring asialo 1-acid
glycoprotein (AsAGP).
In the method for detecting a hepatic disease of
the present invention, test samples can be analysed on
a microplate through a sandwich enzyme immunoassay, an
enzyme immunoassay onto an diagnostic strip for
immunochromatography and various types of enzyme
immunoassay. Especially, the enzyme immunoassay onto
diagnostic strip fur immunochromatography is the most
convenient among these methods.
Preferably, the monoclonal antibody utilized in
the diagnostic method of the present invention is a
subclass type IgGI as described above. More preferably,
the monoclonal antibody is produced from the mouse
hybridoma cell line of the present invention deposited
with accession number KCTC 10261 BP. Besides, as a
lectic, RCA recognising asialo 1-acid glycoprotein is
9

preferable to be utilized.
The present invention provides a diagnostic scrip
for immunochromatography which comprises a monoclonal
antibody binding only with asialo 1-acid glycoprotein
and excluding heptoglobin and 2-macroglobulin; and
lectin RCA recognizing asialo glycoprotein; measure the
concentration of asialo 1-acid glycoproteir. in a test
sample; and diagnose a liver disease rapidly and
easily.
Preferably, the present invention provides the
diagnostic strip for immunochromatography which
includes the monoclonal antibody As 16.85 deposited
with the accession number KCTC 102 61 BP and RCA as a
lectin. The diagnostic strip for immunchromatography of
the present invention comprises glass fiber (GF)
tnerobrane coated with micro-particles such as gold-
colloid conjugated with monoclonal antibody;
nitrocellulose membrane (NC) in which RCA band is lined
as a diagnostic line and an monoclonal antibody band as
a standard line; a sample pad absorbing test sample
solution; an absorbent pad discarding non-reactive
substance; and an adhesive plastic backing for mounting
the above-mentioned members.
The diagnostic strip for immunochroniasography of
the present invention is mounted in due turn,
preferably NC membrane, GF membrane, a sample pad and
absorbent pad are partially overlaid onto the adhesive
10

plastic backing to transfer substance smoothly by
capillary reaction.
The diagnostic strip for immunochromatography can
be prepared by the conventional procedure. Preferably,
the diagnostic strip of the present invention can be
manufactured to a cassette type or a stick type.
Besides, the test sample is preferable to be
blood or serum and 10-fold diluted by using elution
buffer and the elution buffer is preferable to be 50 mM
borate buffer containing 5% sucrose, 1% bovine serum
albumin or 1% Triton X-100.
The diagnostic strip for immunocrhromatography of
the present invention is used to detect a liver disease
as follows: when a test sample is diluted and dropped
onto the sample pad, both the standard line and
diagnostic line are colored on the atrip after 3-5
minutes. SO that the sample be judged according to
colors whether positive for hepatic disease or not.
Precisely, if Ab-gold conjugate is adopted as a
microparticle, the positive sample including asialo 1-
acid glycoprotein reveals red color both on the
standard line and the criteria line after a test and
the negative sample including asialo 1-acid
glyeoprotein in a normal level (not a patient of liver
disease) reveals red color only on the standard line.
As described above, the diagnostic strip for
immunochromatography of the present invention can
11

detect asialo 1-acid glycoprotein to reach a cutoff
value, sbout 1.50 g/ml, which can diagnose a liver
disease early and monitor its prognosis and treatment
if the level of asialo 1-acid glycoprotein present in
patients of liver cirrhosis and liver cancer continued
to be evaluated,
Furthermore as illustrated above, the monoclonal
antibody which reacts only with asialo 1-acid
glyeoprotein and excludes heptoglobin and 2-
macroglobulin and the diagnostic atrip for
immunochromatagraphy by using the same can inform the
test result rapidly and conveniently so that liver
diseases can be diagnosed easily. Therefore, it is
expected to contribute to the prevention and treatment
of liver diseases
EXAMPLES
Practical and presently preferred embodiments of
the present invention are illustrated as shown in the
following Examples.
However, it will be appreciated chat those
skilled in the art, on consideration of this
disclosure, may make modifications and improvements
within the spirit and scope of the present invention.
Separation and purification of asialo 1-
12

acid glycoprotein (AaAGP)
1-acid glycoprotein (AGP) was separated and
purified from asialo glycaprotein contained in human
blood plasma as follows.
2 NIH unit of thronbin was added to 20 0 ml of
human blood plasma, stored at 37 C for 2 hours and at
4C overnight and centrifuged to remove blood clot. The
serum prepared above was dialyzed by using 0,05M of
sodium acetate buffer (pH 4.3), loaded onto DEAE column
previously equilibrated by using the same buffer, and
then eluted through linear concentration gradient after
0.05 M of sodium acetate buffer (pH 4.3) and 0.1 M of
sodium acetate buffer (pH 4.3) were mixed. Afterward,
the optical density (OD) was measured at 280 nm and the
data was illustrated in FIG. 1. The fractions including
AGP and other proteins were collected, mixed with
antmonium stilfate to 0.5 g/ml and centrifuged to
precipitate proteins. The resulting supernatant was
mixed again with ammonium sulfate bo 0.18 g/ml to
precipitate proteins and the pellet was dissolved in a
small amount of distilled water (D, W.), dialysed
sufficiently by using D. W. and then lyophilieed.
40 mg of AGP separated above was hydrolyzed at
80C for 2 hours by using 6 ml of 0.1 N sulfate
solution, neutralized by using 1 N of sodium hydroxide
and then dialysed by using 0,01 N of phosphate buffer
13

(pH 7,4) . The desialylaced 1-acid glycoprotein (AsAGP)
prepared in the above procedure was loaded onto
Sephadex G-200 column, filtrated by using 0.01 M of
phosphate buffer (pH 7.4) and calculated through OD
value at 280 nm and finally the fractions including
proteins were collected. The result was illustrated in
FIG. 1: lane 1 is the standard marker of protein
molecular weight; lane 2 is 1-acid glycoprotein; lane
3 and 4 are asialo 1-acid glycaprotein (AsACP).
Preparation of monoclonal antibody against
asialo 1-acid glycoprotein
(1) Immunization of mice
In order to obtain an, immunised mouse essential
to prepare a hybridoma cell line producing a monoclonal
antibody against asialo 1-acid glycoprotein, asialo
1-acid glycoprotein as an antigen was suspended well
by using Titer-MAX, adjusted to 50 g/50 ml of
concentration and then injected into peritoneal cavity
of Balb/c mice aged 6-8 weeks. After 2 weeks, the
same amount o£ antigen mentioned in the first injection
was mixed with Titer-MAX and injected repeatedly onto
the same site. Through the same procedure, the antigen
was injected again after 7 days and repeatedly injected
after 3 weeks. Afterward, the small amount of blood was
collected from a tail of mouse and examined to evaluate
14

a titer.
(2) Cell fusion
In order to perform cell fusion for preparing a
hybridoma cell line, mice were immunised with asialo
1-acid glycoprotein antigen mentioned above and spleen
cells and myeloma cells were collected front the mice.
Then, 103 of spleen cell and 107 myeloma cell (SP2/0)
were washed sufficiently, mixed together and 1 ml of
PEG 1500 was poured for about 1 minutes, stirred
slightly for about 1 minutes. Afterward, 9 ml of RPMI
medium was added for about 3 minutes to reach 50 ml of
final volume as stirred slowly. The cell suspension was
centrifuged to collect the cell pellet, suspended Again
to 1-2 X 103 cells/ml by using HAT medium, poured into
a 96-well microplate in 0.2 ml volume per well, and
then incubated at 37 C in CO2 incubator,
(3) Screening of hybridoma cell producing
monoclonal antibody
In order to select a hybridoma cell specific for
asialo 1-acid glycoprotein, ELISA method was tried by
exploitrig a microplate coated with asialo 1-acid
glycoprotein as follows. Precisely, asinlo 1-acid
glycoprotein antigen was put into a microplate in 100
1 (1 g/ml) per well to coat the surface and washed
off to remove antigens not reacted. The culture medium
15

of hybridoma cell was poured to each well in 100 1,
reacted for 2 hours and washed off by using Tween 20
phosphate buffer (PBST) Co remove the culture medium
not reacted. Afterward, goat anti-mouse IgG-horseradish
peroxidase (HRP) was added, reacted for an hour at room
temperature and washed off by using PBST solution.
Then, ortho-phenylenediamine (OPD) was utilized, as a
substrate of peroxidase, reacted and examined to
measure OD value at 490 nm with ELISA reader.
As a result, the cell line secreting antibodies
highly binding to asialo 1-acid glycoprotein antigen
was first screened and repeatedly selected to separate
the hybridoma cell secreting a monoclonal antibody
specific for asialo 1-acid glycoprotein antigen. The
resulting cells were treated by the limiting dilution
to become a monoclone and the hybridoma, cell line
producing a monoclonal antibody was sorted again.
Afterward, 2 clones having the highest titer such as
asialo 1-acid glycoprotein 1 and asialo 1-acid
glycoprotein 2 were collected, tried to measure the
titer of supernatant in culture medium through ELISA
method and then the subunit type of the monoclonal
antibody Was analyzed by the double immunodiffusion,
Consequently, the antibodies secreted from the clones.
of the present invention were identified as IgGI and
IgM reapectively.
The cell line secreting IgGI has been named aa AS
16

16.89 said deposited to Korea Research Institute of
Bioecience and Biotechnology, Gene Bank in May 24, 2004
(accession number KCTC 10261 BP) .
(4) Large scale production and purification of
monoclonal antibody
In order to produce a monoclonal antibody from a
hybridoma cell in a large scale, 0.5 ml of pristane was
injected into peritoneal cavity of Balb/c mice. After 1
week, each cell line obtained in the above procedure
(3) was injected to the experimental mice in 5 x l06
cells per mouse and then peritoneal fluid was collected
from the peritoneal cavity if swollen. The peritoneal
fluid was centrifuged at 12,000 rpm to collect cells
and the resulting supernatant was filtrated through
protein G column to separate and purify the monoclonal
antibody against asialo 1-acid glycoprotein and, also
stored at -20 C for the next experiments.
(5) Identification of specificity of monoclonal
antibody by western blot
In order to elucidate the property of monoclonal
antibody specific for asialo 1-acid glycoprotein, the
monoclonal antibody against asialo 1-acid
glycoprotein purified through the above-mentioned
procedure (4) was identified by performing SDS-
polyacrylamide gel electrophoresis and western blotting
17

[See FIG 2) . The western, blotting was accomplished by a
typical procedure in the prior arts, in FIG. 2, lane 1
is a standard marker of protein molecular weight; lane
2, 1-acid glycoprotein; Lane 3, asialo 1-acid
glycoprotein; lane 4, 2-macroglobulin; and lane 5,
heptoglobin.
(6) Identification of specificity of monoclonal
antibody by enzyme immunochemical assay
Asialo 1-acid glycoprotein, heptoglobin, 2-
macroglobulin antigen were added to 96-well microplate
in 100 1 (1 g/ml) per well respectively, attached on
the surface, reacted for 2 hours with the monoclonal
antibody against asialo 1-acid glycoprotein as added
in 100 1, and washed off by using phosphate buffer-
Tween 20 (PBST) to remove the culture medium not
reactfed. Further, goat anti-mouse IgG-HRP was added,
reacted for an hour at room temperature and washed
sufficiently by using PBST solution. Afterward, ortho-
phenylenediamine (OPD) was added as a substrate of
peroxidase and reacted so as to measure OD value at 490
nm with ELISA reader. As a result, it is confirmed that
the monoclonal antibody against asialo 1-acid
glycoprotein purified above be the monoclonal antibody
specific for asialo 1-acid glycoprotein. {See FIG. 3} .
In FIG. 3, AGP depicts 1-acid glycoprotein and AsAGF,
asialo 1-acid glycoprotein of the present invention.
18

Detection of liver disease by diagnostic
kit for immunochromatography
The diagnostic kit for immunochromatography of
the present invention which can measure the
concentration of asialo 1-acid. glycoprotein in a test
sample by the immune reaction between, the monoclonal
antibody. As 16.89 specific far asialo 1-acid
glycoprotein produced above and asidlo 1-acid
glycoprotein in blood was manufactured and applied in
clinical fields as follows.
(1) Preparation of diagnostic kit for
immunochromatography
The diagnostic kit for detecting asialo 1-acid
glycoprotein in blood is composed of following
components as described below.
A. monoclonal antibody (As16.89)
specific for asialo 1-acid
glycaprotein fixed onto solid
carrier such as microplate
19

B. lectin. (RCA) -horseradish peroxidase
(HBP) against asialo 1-acid
glycoprotein
C. sample dilution buffer (1% BSA/PEST)
D. enzyme eubstrate solution (OPD)
E. washing buffer (PBST)
F. standard solution of asialo 1-acid
glycoprotein
G. stopping buffer
(2) Meagurement
a. The standard solution (1 g/ml) of asialo 1-
acid glycoprotein was poured in 100 1 respectively to
mieroplate on which the monoclonal antibody As 16.89
was fixed. Experiment samples, including 40 normal
persons, 155 patients without liver disease, 36
patients of acute hepatitis, 272 patients of chronic
hepatitis (CH), 230 patients of liver cirrhosis, 72
patients of hepatocarcinoma (HCC) proceeded from liver
20

cirrhosis, were obtained from St. Mary Hospital,
Catholic Medical School. The blood sample was diluted
to 1 : 10 ratio by using the dilution buffer, allotted
onto each well of microplate in 100 1 and incubated
for 120 minutes at room temperature.
b. After reacted, the washing buffer was added
in 100 1 per well and washed three times repeatedly.
c. The lectin (RCA)-HRP diluted by the process
for the preparation was allotted onto each well of
microplate and reacted for 60 minutes at room
temperature.
d. Step b was repeated.
e. The enzyme substrate solution (OPD) was
allotted onto each well of microplate above.
f. The stopping buffer was added on each well in
10 0 1 to stop the ensymatic reaction.
g. OD value was measured at 490 nm with RLISA
reader.
As a result, the content of asialo 1-acid
glycoprotein in blood was evaluated through the above-
21

mentioned procedure as illustrated in Table 1.
22

chronic hepatitis, liver cirrhosis and hepatocarcinema,
with liver cirrhosis, the blood level of AsAGP was
evaluated to average 1.33 g/ml, 1.63 g/ml, 3.12 g/ml
and 3.64 g/ml respectively. The concentrations be
remarkably distinguished from those of standard group,
even if the ratio of sample over 1.5 g/ml of AsAGP be
25%, 36%, 72% and 82% respectively, Therefore, it is
verified that this test be effective upon the diagnosis
of hepatic diseases.
Hence, according to data demonstrated above, the
cutoff value for diagnosing a liver disease was decided
to be 1.50 g/ml when the monoclonal antibody As 16.39
of the present invention was used for the enzyme
immunoassay.
Preparation of diagnostic strip for
immunochromatography.
(1) Preparation of monoclonal antibody-gold
conjugate
The monoclonal antibody specific for asialo 1-
acid glycoprotein selected in the present invention was
added in 15 g/ml to the colloidal solution of gold
particles, reacted for 2 hours at room tempsrature and
rotated- Then, 10% of BSA was blended in 1/10 volume to
become 1% of concentration and again reacted for an
hour to prepare Ab-gold conjugate. Afterward, the
25

resultant was centrifuged at 12,000 rpm for 40 minutes
to discard the supernatant solution and 2 mM of borate
buffer was added to wash off Ab-gold conjugate. The
resultant wag washed repeatedly, three times and
finally 2 mM of borate buffer containing 1% BSA was
added in about 1/10 volume of gold solution and
suspended. After measured with UV spectrometer, the OD
value at 530 nm was adjusted to 3.00 by dilution.
(2) Sample pad
The sample pad is a member absorbing test sample
and is made of cellulose materia1 for this purpose.
(3) Glass fiber (GF) metobrane
The glass fiber is a component on which AsAGP in
tesc sample and the monoclonal antibody prepared by the
process of the present invention are reacted. The GF
membrane was coated with gold colloid particle-As 16.89
on the surface and prepared as follows.
The monoclonal antibody produced from hybridoma
call line. As 16.39 of the present invention was
conjugated onto the membrane as illustrated in step
(1).
GF membrane (1.0 cm X 0.7 cm) purchased from
Millipore Co. Ltd, was soaked by using 20 mM sodium
borate buffer, sprayed uniformly by using gold colloid
particle-As 16.89 onto the surface, dried at 37 C and
26

thus made to GF membrane pad for immunochromatography
(also conjugate pad) on which a monoclonal antibody
conjugated with a coloring particle is fixed.
(4) Nitrocellulose (NC) membrane and lining
Nitrocellulose membrane purchased from Millipore
Co. Ltd. was cut to a proper size (0.7 cm X 5 cm) ,
lined at a point of about 2.4 cm from the bottom of
plaetic backing with goat anti-mouse IgG as a standard
line and at a point of 2.7 cm as a criteria line for
detecting asialo 1-acid glycoprotein by using RCA
purchased from EY Lab, and then dried to complete the
NC membrane.
(5) Absorbent pad
Cellulose membrane was used to absorb substance
not reacted in a test sample after an immune reaction.
and to transfer the sample solution through capillary
reaction.
(6) Preparation of diagnostic strip for
immunchromatography.
As illustrated in FIG. 4a and FIG. 4b, the above-
mentioned members were mounted onto the adhesive
plastic backing. Precisely, NC membrane, GF membrane,
sample pad and absorbent pad were arranged in due turn,
overlaid evenly in about 0.1 cm portion and fixed by
27

adhesion in order to transfer substance smoothly
through capillary reaction,
Analysis of asialo 1-acid glycsprotein by
using diagnostic strip for immunochromatography
Sarum sample was diluted to 1 : 10 ratio by using
elution buffer (such as 50 mM borate buffer containing
5% sucrose, 1% bovine serum albumin or 1% Triton X-100)
and added in 60 - 70 I volume onto the sample pad of
diagnostic atrip for immunochramatography prepared in
Example 4 so as to examine the standard line and the
criteria line after 3 - 5 minutes whether colored or
not.
FIG. 5 depicts the result from the cassette type
strip for immunochramatography. NO. 1 is the standard
dropping the mixture of 1-acid glycoprotein (AGP),
heptoglobin and 2-macraglobulin; NO. 2, the diagnostic
strip in the serum of normal person; NO. 3 -NO- 6, the
strip id 1.5 g/ml, 2.0 g/ml, 3.0 g/ml and 4.0 g/ml
of asialo 1-acid glycoprotein respectively. As
illustrated in FIG. 5, in NO. 3, the line is obscure;
and in MO. 4 - NO- 6, red colors are clear both on the
standard line (upper) and the criteria line (lower).
The diagnostic strip for immunochramatography of
the present invention is observed that only the
standard line become red in the test sample of normal
28

person (negative) under the cutoff value, 1.50 g/ml of
asialo 1-acid glycoprotein since antigen-antibody-
enzyme conjugate complex wag not formed. On the other
hand, when asialo 1-acid glycoprotein level increased
bo more than 1.50 g/ml due to a liver disease, both
the lines become red, in which asialo 1-acid
glycoprotein was absorbed on the sample pad, reacted
with gold particle-monoclonal antibody As 16.89
conjugate on GF membrane to form an immune complex,
transferred onto NC membrane through, capillary reaction
and conjugated with RCA in the criteria line on NC
membrane to make gold precipitate. Furthermore, the
criteria line can be varied in thickness and color
density, depending upon the concentration of asialo 1-
acid glycoprotein. Therefore, it is possible to predict
the severity of liver disease by the concentration of
asialo 1-acid glycoprotein in the test sample.
INDUSTRIAL APPLICABILITY
As illustrated above, the monoclonal antibody
specific for asialo 1-acid glycoprotein and the
diagnostic strip and the diagnostic kit for
immunochramatography by using the same of the present
invention can be utilized to evaluate asialo 1-acid
glycoprotein present in blood sample rapidly and
easily. Therefore, the monoclonal antibody and the
29

diagnostic device of the present invention can monitor
the severity, the treatment and Che prognosis of liver
diseases efficiently.
Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the
foregoing description may be readily utilized as a
basis for modifying or designing other embodiments for
carrying out the same purposes of the present
invention.
Those skilled in the art will also appreciate
that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the
appended claims.
30

What is claimed is:
1. A monoclonal antibody which reacts only with
asialo 1-acid glycoprotein. and excludes heptoglobin
and 2-macroglobulin.
2. The monoclonal antibody according to claim 1,
which is a subclass type IgGI.
3. A cell line which produces in a large scale a
monoclonal antibody reacting only with asialo 1-acid
glycoprotein and excluding heptoglobin and 2-
macroglobulin.
4. The cell line according to claim 3, which is
prepared by fusing a spleen cell and a myeloma cell
extracted from a mouse immunised with asialo 1-acid
glycoprotein.
5. The cell line according to claim 4, which
produces the subclass type IgGI monoclonal antibody
specific for asialo 1-acid glycoprotein and is
deposited to Korea Research Institute of Bioscience and
Biotechnology, Gane Bank in May 24, 2004 (accession
number KCTC 10349 BP) under Budapest Treaty.
6. A method for diagnosing a liver disease in which
31

a monoclonal antibody which reacts only with asialo 1-
acid glycoprotein and excludes heptcglotin and 1-
macroglobulin; and lectin RCA (Ricinua communis
agglutinin) recognizing asialo glycoprotein are reacted
with a test sample to measure the amount of asialo 1-
acid, glycoprotein (AsAGP).
7. The method for diagnosing a liver disease
according to claim 6, in which the monoclonal antibody
is the subclass IgGI produced from the mouse cell line
deposited with the accession number KCTC 10261 BP.
8. The method for diagnosing a liver disease
according to claim 6, in which the test sample is blood
or serum.
9. An diagnostic strip for immunochramatography
which comprises a monoclonal antibody reacting only
with asialo 1-acid glycoprotein excluding heptoglobin
and 2-macroglobulin; and RCA recognizing asialo
glycoprotein as a lectin; and reacts a test sample at
over 1.50 g/ml of asialo 1-acid glycoprotein to
detect a liver disease.
10. The diagnostic strip for immunochromatography
according to claim 9, in which the monoclonal Antibody
is As 16.89 deposited with the accession number KCTC
32

10261 BP.
11. The diagnostic strip for immunochramatography
according to claim 9, in which the monoclonal antibody-
is conjugated with a micro-particle such as colloid
type gold particle.
12. The diagnostic strip for immunochramatography
according to claim 11, which comprises (1) glass fiber-
(GF) coated with, micro-particles conjugated with the
monoclonal antibodys (2) nitrocellulose membrane (NC)
including a standard line and a diagnostic line to
detect a glycoprotein; (3) a sample pad for a test
sample; (4) a absorbent pad for non-reactive substance;
and (4) an adhesive plastic backing for imountiag above-
mentioned naembers.
13. The diagnostic strip for immunochromatography
according to claim 12, in which NC meaibrane, GF
membrane, a sample pad and a absorbent pad are
partially overlaid on said adhesive plastic backing in
due turn to transfer substance by capillary reaction
and an RCA band as a diagnostic line and an antibody
band as a standard line are separated in some interval.
14. The diagnostic- strip for immunochramatography
according to claim 9, in which said beet sample is
33

20. The diagnostic kit for immunochramatography
according to claim 19, in which, the monoclonal antibody
is As 16.89 deposited with the accession number KCTC
10261 BP.
35

The present invention relates 10 a method for diagnosing; a livar disease rapidly in an early stage. More particularly,
the present invention relates to a monoclonal antibody aganist asialo a 1-acid glycopreting; a method for diagnosing a liver dis-
ease which XXX asialo a I-acid flycopratein in a test sample by asing said monoclonal antibody; and an diagnostic strip for
XXX composed of said monoclonal antibody against XXX a l-acid glycoprotein and Rictons community aggln.
tinin ()RCA). The diagnostic devlee of the present invention is convanicnt to measure the concentration of asialo a l-acid glycoprotein
and to diagnose a liver disease rapidly.

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Patent Number

219325

Indian Patent Application Number

01296/KOLNP/2005

PG Journal Number

18/2008

Publication Date

02-May-2008

Grant Date

30-Apr-2008

Date of Filing

05-Jul-2005

Name of Patentee

NEOBIODIGM CO., LTD.

Applicant Address

52, EOUN-DONG, YUSEONG-GU, DAEJEON 305-333, REPUBLIC OF KOREA

Inventors:

#	Inventor's Name	Inventor's Address
1	CHUNG, TAI-WHA	SHINDONG-A APT. 12-1301, YONGJEON-DONG, DONG-GU, DAEJEON 300-766, REPUBLIC OF KOREA
2	SONG, EUN-YOUNG	HANSUNG APT. C-1001, YEOUIDO-DONG, YOUNGDEUNGPO-GU, SEOUL 150-010, REPUBLIC OF KOREA
3	KANG, JI-HYUN	HONERS VILL 1013 HO, 1380-1, DUNSAN 1-DONG, SEO-GU, DAEJEON 302-121, REPUBLIC OF KOREA
4	KIM, KYOUNG-A	TAEJONG ARTVILLA GA-201, BIRAE-DONG, DAEDEOK-GU, DAEJEON 306-030, REPUBLIC OF KOREA
5	LEE, EUN-YOUNG	ARTVILLA 205 HO, 101-2, EOUN-DONG, YUSEONG-GU, DAEJEON 305-333, REPUBLIC OF KOREA
6	CHOE, YONG-KYUNG	HANSOOP APT, 103-1504, YONGJEON-DONG, DONG-GU, DAEJEON 300-768, REPUBLIC OF KOREA

PCT International Classification Number

C07K 16/18

PCT International Application Number

PCT/KR2003/002860

PCT International Filing date

2003-12-27

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10-2002-0084834	2002-12-27	Republic of Korea