METHOD FOR ASSAYING BASIC FETOPROTEIN AND REAGENT THEREFOR

Abstract: A method for assaying basic fetoprotein by a sandwich method with the use of two antibodies is characterized in that monoclonal antibasic fetoprotein antibodies which react with antigenic determinants of the basic fetoprotein different from each other are selected as said two antibodies.

Description: METHOD FOR ASSAYING BASIC FETOPROTEIN AND REAGENT THEREFOR This invention relates to a method for assaying basic fetoprotein and a reagent therefor. Basic fetoprotein (BFP), which is a basic protein having a molecular weight of 73,000 and an isoelectric point of 9.3 and migrates in the Y-giobulin region in electrophoresis, has bean found by one of,the inventors in serum, intestinum and brain tissues of a human fetus. It has been particularly called basic fetoprotein since it is basic in contrast to the known acidic fetoprotein Furthermore one of the inventors has established a radioimmunsassay system of said protein and found that assay of said protein in serum would help to diagnose cancer and to judge its condition and therapeutical effect thereon. In particular, it has been found that said fetoprotein is useful to examine the presence of cancer, differing from &alpha;-fetoprotein which is useful to diagnose cancer of a particular organ. These prior findings are described in the following references (1) to (7) which are cited as referential information of the present invention. (1) Ishii, M. et al. : Studies on feto-neoplastic antigen. Proceedings of the 34th General Meeting of Japan Cancer Society,! p 173 (1975) (2) Ishii, M.: Studies on novel basic fetoprotein found in various malignant tumors. Igaku no Ayumi, 100 (3), 344 - 346 (1977). (3) Ishii, M.: Basic fetoprotein. Igaku no Ayumi, 106 (5), 275 - 281 (1978). (4) Ishii, M : A new carcinoembryonlc protein characterized by basic property. Scandinav. J. Immunol., 8 (Suppl 8), 611 - 620 (1978). (5) Ishii, M.: Characterization of basic fetoprotein and clinical usefulness of BFP for immunodiagnosis of human cancer. Carcino-Embryonic Proteins. Chemistry, Biology, Clinical Application, Vol. 1, Lehmann, F. - G. (ed.), Elisevier/North-Holland Biomedical Press, Amsterdam, 333 - 340 (1979). (6) Ishii, M. ; Nishimura, K., Hattori, M., Kanda, Y. and Ishihara, A.: Postoperative surveillance in patients with stomach cancer and monitoring of immunoand polychemotherapy in patients with leukemia by basic fetoprotein. Carcino-Embryonic Proteins. Chemistry, Biology, Clinical Application, Vol. 2 Lehmann, F. - G. (ed.), Elisevier/North-Holland Biomedical Press, Amsterdam, 603 - 606 (1979). (7) Ishii, M.: Clinical usefulness of basic fetoprotein for immunodiagnosis of human cancer. Compendium of A@@ay for Immunodiagnosis of Human Cancer, Development in Cancer Research, Herberman, R.B. (ed.), Vol. 1, Elisevier/North-Holland Biomedical Press, New York, 45 - 50 (1979). Since basic fetoprotein is present in the serum of cancer patients in a trace amount, it is necessary to establish a highly sensitive assay system to determine it. RIA and EIA methods nave been mainly developed therefor. Detailed descriptions of the RIA method are found in the references (3) to (5) as cited above. Detailed descriptions of the EIA method are found in the following references (8) End (9) which are also cited as referential information. (8) Ishii, M et i1: Basic fetoprotein, present clinical function test - enforcement and interpretation thereof. Nippon Rinsho 37, 1536 - 1539 (1979). (9) Ishii, M : Basic fetoprotein: Rinsho Kensa 24 (8), 931 - 936 (1980). Subsequently the inventors have prepared monoclonal antibasic fetoprotein antibodies in order to mass-produce the antibodies used in the RIA and EIA methods and to further heighten the sensitivity of these methods by the use of said antibodies. Preparation and evaluation of said monoclonal antibodies are described in the following reference (10) which is also cited as referen- tial information. (10) Proceedings of the 10th Japan Clinical Immunology Assoc. (Osaka). Preparation of monoclonal antibodies for basic fetoprotein. The reaction specificities for basic fetoprotein of the monoclonal antibodies thus obtained were examined by the EIA and MO methcds. Consequently it was found that there are at least three different antigenic determinants on She basic fetoprotein molecule and that the monoclonal antibodies might be classified into three types corresponding to each antigenic determinant. Examples of monoclonal antibodies corresponding to the three antigenic determinants A, B and C are as follows: antigenic determinant monoclonal antibody A 5C4, 5C5, 5C6, 7D1, K1 B 5B2, 5B3, 5C2 C 5A-2, 5A3, 5D3, 7D3, 8A2, 7B4, 5DE-2, 8A1, 7A5-2, 8A5, 7B6 Under these circumsuances, we have studied to estab lish a highly sensitive method for assaying basic 'eío- protein by a sandwich method with She use of monoclonal antibodies from various viewpoints. As a result, we have found that monoclonal antibodies which react with antigenic determinants different from each other should be combined as the two ant@bodies used in the sandwich method. That is to say, combinations of 5C4 and 5C2 or K1 and 5B3 monoclonal antibodies in the foregoing example may be selected Thus the present invention has been completed to provide a method for assaying basic fetoprotein based on these findings. Accordingly, it is an object of the present invention to provide a highly sensitive method for assaying basic fetoprotein by a sandwich method. The present invention discloses a technique for selecting monoclonal antibasic fetoprotein antibodies which react with antigenic determinants different from each other as the two antibodies used in the sandwich method to thereby achieve the abovementioned object. Now the present invention will be described in detail. As described above, the basic fetocrotein assayed by the method of the present invention is a basic protein which has a molecular weight or 73,000 and an isolectric point of 9.3, migrates in the T-globl n region in electroDhoresis and serves as a tumor marker having a low organ specificity. Monoclonal antibasic fetoprotein antibodies used in the present invention may be prepared, for example, in the following manner. Antibody-forming cells obtained from a BALB/C mouse sensitized with basi@ fetoprotein are fused with myeloma cells, P3-X63-Ag8-U1, by z variation of the method repori2a by Herzenberg et al. Subsequently the antibody titer of the fused cells selected by a HAT medium wns assayed by plate binding assay with 125I 30me the use of basic feGoprotein labeled with cell strains showing high antibody titers are obtained by cloning fused cells which exhibit a high antibody titer and excellent profliferation by limiting dilution. Finally each cell strain thus obtained is transplanted nnto the abdominal cavity of a mouse and its hydroperitoneum thus obtained is salted out with ammonium sulfate, dialyzed and subjected to DEAE-cellulose column chromatography to collect an IgG component, thereby obtaining some monoclonal antibodies available in the present invention. The monoclonal antibodies thus obtained may be classified into three types corresponding to the three antigtPnic determinants of basic fetoprotein by the MO and EIA methods as described below. MO method: Mixtures of all combinations fo two monoclonal antibodies at a ratio of 1 : 1 are prepared. Formation of a sedimentation line and the presence of fusion berween these mixtures and basic fetoprotein are observed by the MO method When an obvious sedimentation line is observed, the onoclonal antibodies consLituting the mixture are classified into different groups which react with antigenic determinants different from each other. On the other hand, when no sedimentation line is observed, they belong to the same group which reacts with an antigenic determinant. When an unobvious sedimcn- ration line makes it impossible to judge definitely, the following EIA method may be employed. EIA method: The procedure of Example 1 is followed except that two monoclonal antibodies selected arbitrarily are used instead of the K1 and 5C2 antibodies to prepare reagents of every combination of the monoclonal antibo- dies. Coloration velue (OD405nm) is determined by EIA operations. When coloration is observed, the monoclonal antibodies constituting the combination are classified into different groups which react with antigenic determinants different from each other On the contrary when no coloration is observed, they belong to the same group which reacts with an antigenic determinant. Thaws the monoclonal antibodies can be classified into three groups corresponding to the three dilferent types of antigenic determinants of basic fetoprotein by the MO and EIA methods Now the method for assaying of the present invention will be described in detail. The method for assaying of the preset invention is a sandwich method with the use of two antibodies and carried out by taking advantage of either enzyme immunoass2y or r2dioimmunoassay. Usual procedures of these assays are well known per se. Thus the method of assaying of the present invention by enzyme immunoassay may be carried out as follows. The whole assay system consists of 2 solid phase, an antigen for coating the solid phase (the first antigen), basic fetoprotein (a standard antigen and test serum), a marker antibody (the second antibody), an enzyme and a substrate. A well of a microtiter-plate for enzyme immunoassay may be used as the solid phase. Prior to the assay, one of the monoclonal antibodies of the present invention is selected arbitrarily as the antibody for coating the solid phase (i.e. the first antibody), dissolved in a 0.1 M phosphate buffer physiological saline (pH 7.2) to give a protein concen- tration of OD280nm of 0.050, stored, for example, in a polystyrene well for enzyme immunoassay and allowed to stand overnight at 4 C to thereby coat the surface of the solid phase with the first antigen The standard antigen may be prepared by purifying hydroperitoneum obtained from a patient suffering from hepatoma to prepare purified basic fetoprotein and diluting it to a certain concentration with a standard antigen diluent A preferred marke- antibody (i.e. the second antibody) is a monoclonal antibody of She present invention which reacts with an antigenic determinant different from the one with which the first antibody reacts. Example of available enzymes are alkali phcsphatase, glucose oxidase, peroxidase and ss-galactosidase. Prior to the assay, the marker antibody may be bonded to the enzyme with a bonding agent such as gluiaraldehyde to form a conjugate which may be used as a part of the reagent of the present invention. The substrate may be ç bitrarily selected depending on the enzyme to be used. For example, p-nitrophen5yl phosphate may be used when alkali phoscha- tase is selected as the enzyme. The assay may be carried out in a conventional manner of enzyme immunoassay. Thus, as shown in Examples as described below, it may be carried out by introducing a standard antigen or test serum into a cuD coated with the first antibody to incubate, subseauently adding an enzyme marker antibody (e.g. the second antibody/ alkali phosphatase conjugate) to incubate, finally adding the substrate (e.g. p-nitrophenyl phosphate) to incubate and detenmining the amount of a decomposed substrate with a spectrophotometer. The first antibody used for coaxing the solid phase may be either a single monoclonal antibody or a mixture of several monoclonal antibodies. That is to say, it is not the deGermining factor in the present invention whether the first and the second antibodies consist of a single or several monoclonal antibodies so long as the first and the second antibodies react with antigenic determinants different from each other. As shown in Experimental. Example as described below, the method for assaying of the present invention is characterized in that similar effects may be obtained in determining the coloration value of the standard antigen and basic fetoprotein value (BFP value) in normal human serum to those obtained by using polyclonal antibodies. The reagent of the present invention is directly available for said method for assaying of the present invention to achieve the same object as thai of the method. Accordingly said reRgenb may be used ill the sandwich method with the use of two antibodies by taking advantage of either enzyme immunoassay or radioimmunoassay. Now an example of an embodiment of the reagent off the present invention used in enzyme immunoassay will be illustrated. The reagent of the present invention is a set which essentially consists of an antibody for coating a solid phase (i.e. the first an zbody) and a marker antibody (i.e. the second antibody) optionally with other component(s) selected from a solid phase, a standard antigen, an enzyme and a substrate. When a solid phase is included in the set, said solid phase may be coated with the first antigen. When an enzyme is included in the set, said enzyme may be bonded to the second antibody to thereby form a conjugate. These seis may also be referred -to as the reagent of the present invention. Furthermore, other agents suitable for carrying out the assay conveniently, such as an antigen diluent, a reaction diluent, a reaction terminating solution, a substrate solution or a buffer solution, may be included in the set without limiting the present invention. Brief Description of the Drawing: Fig. 1, which corresponds to Fig. 1 described in Experimental Example 1, is a graph showing a correlation between BFP values each determined by assaying with the use of monoclonal antibasic fetoprotein antibodies and by assaying with the use of polyclonal antibasic fetoprotein antibodies. To illustrate the effect of the present inveYltion, the following Experimental Example will be given. Experimental Example 1 Saddle: A solid phase first antibody and an enzyme marker antibody were prepared from monoclonal antibasic feto protein antibodies in a similar manner as described in Example 1 as shown below. On he other hand, another solid phase first 2nti- body and an enzyme marker antibody were prepare from polyclonal antibasic fetoprotein antibodies in ther following manner. Serum which was separated from blood obtained from a rabbit immunized with basic fetoprotein thrice was salted out and purified by means of affinity column chromazography using basic fetoprotein to thereby obtain polyclonal antibasic fetoprotein. Subsequently a porTion whereof was dissolved in a 0 1 M PB (pH 7.2) to give a protein concentration of OD280nm 1 05 poured into a well of a microtiter plate for enzyme immunoassaY and alloweÅa to stand overnight. The liquid was removed and the residue was washed with demineralized water and dried by a cup drier to whereby obtain a solid phase first antibody. Another portion of polyclonal antibasic fetoprotein was mixed with alkali phosphaase --o give a concentration of appro- ximately 1 mg/mS and a 2 % glytaraldehyde was added slowly to give a final concentraiion of 0.2 %. After allowing to react; for 30 min at room temperature, the reaction mixture was dialyzed against a 0.05 M Tris hyarochloride buffer solution and 100 % NRS was added to give a final concentration of 25 %. Aster adjusting to an optimum concentration with a 0.05 M Tris hydrochloride buffer solution (pH 8.0) which contained 10 % of NRS, 1 mM of MgC# and 0.9 % of NaC#, an enzyme marker antibody was obtained Reaction diluents prepared in a similar manner as described in Example 1 were used in a system with the use of monoclonal aitibasic fetoprotein antibodies, while reaction diluents prepared in the following manner were used in a system with th the use of polyclonal anti- basic fetoprotein antibodies. That is to say, normal rabbit serum, trisodium ethylenediaminetetraacetate, and sodium chloride were added to a 0.1 M carbonate buffer solution (pH 9.0) to give concentrations of 12 % (20 % when a standard antigen was diluted), 10 mM and 0.9 %, respectively. A substrate solution and a reaction terminating solution were prepared in the same manners as describeÅa in EXample 1. 180 examples of normal human serum and 19 examples of that of cancer patients were prepared as specimens. Method: j BFP values (ng/mS) of all specimens were determined by assaying with the use of monoclonal antibasic fetoprotein antibodies and polyclonal antibasic fetoprotein antibodies respectively in the following manner; A specimen and a standard antigen having a known concentration were diluted ll-fold with a reaction diluent and poured into wells which had been washed previously in 100 # portions and incubated for one hour at v7 C. After washing wit; ; distilled water, 100 # of an enzyme marker antibody of the optimum concentration was added and incubated for one hour at 37 C. After rehashing with distilled water, 100 # of a substrate solution was added and incubated for one hour at 37 C. Subsequently 100 m# of a 1 N NaOH was added to stop the reaction and the optical density was determined at 405 nm (OD405hm) to calculate the BFP value. Results: Fig. 1 shows the result. In Fig. 1, the abscissa refers to the BFP value determined by the assay with the use of monoclonal antibasic fetoprotein antibodies, while the ordinate refers to the BFP value dei;ermined by the assay with the use of polyclonal antibasic fetoprotein antibodies. Accordingly, Fig. 1 is a graph showing a correlation between these BFP values. The equation of the regression line in the graph :s y = 1.016 x + 0.06 and the correlation coefficienr. &gamma; is 0.981. Thus these BFP values exhibit an excellent correlation with each other, which suggests that the method of the present invention may bring about a similar result to that obtained by assaying with the use of polyclonal antibodies. To further illustrate the present invention, the following examples will be given. Example 1 To 5 m# portions of mouse hyaroperitoneum which contained monoclonal antibodies K1 and 5C2 each reacting with antigenic determinants of basic fetoprotein different from each other, a saturated ammonium sulfate solution (4.05 M) was added to give a final concentration of 1.8 M and stirred for two hours at room temperature. Each mixture was centrifuged at 12,000 rpm for 20 min to thereby separate precipitate from supernatant. The precipitate was dissolved in 3 m# of a 0.01 M PBS (pH 8.0) and dialyzed against the buffer solution used to dissolve the precipitate. The di21yzate was poured into a DEAE-cellulose column to elute the IgG component with a 0.1 M PB (pH 8.0). The eluted IgG component was concentrated by a collodion pack (MW 75,000) to prepare Kl and 5C2. The Kl was dissolved in a 0.1 M PS (pH 7.2) to give a protein concentration of OD280nm of 0.05 and the solution was poured into a well of a microtiter plate for enzyme immunoassay and allowed to stand overnight. After removing the liquid, the residue was washed with demi neralized water and dried by a cup drier to prepare a solid phase first antibody. On the other hand, the 5C2 and alkali phosphatase (5000 u/m#) were each dialyzed against a 0.075 M PBS. The IgG component was mixed with alkali phosphatase to give a concentration of approximately 1 mg/m# and a 2 % glutaraldehyÅae was added slowly with a micro syringe to give a final concentration of 0.2 %. The mature was allowed to react at room temperature for 30 min and dialyzed against a 0.05 M Tris hydrochloride buffer solution (pH 8.0). After adding 100 % NRS to give a final concentration of 25 % and calibrating, the resulting solution was lyophilized to prepare an enzyme marker antibody. Separately reaction diluents, a substrate solution, a reaction terminating solutian and standard antigen iluents were prepared in the Hollowing manner. Norm21 rabbit serum, normal mouse serum (inactivated at gi6 C for 30 min), trisodium ethylenediaminetetra- acetate, sodium chloride and sodium triazide were added to a 0.05 M Tris hyarochloride buffer solution (pH 8.0) to give concentrations of 11.8 %, 2 %, 10 mM, 0.15 M End 0.1 % resDectively, to thereby prepare reaction c iluents, p-Nitrophenyl phosphate was added to a 0.05 M carbonate buffer solution (pH 9.4) containing 2 mM of EgCI'2 to give a concentration of 4 mg/mQ to thereby prepare a substrate solution. 1 N-NaOH was prepared as a reaction terminating solution. Normal mouse serum (inactivated at 50 C for 0 min), trisodium ethylenediaminetetraacetate, sodium chloride and sodium triazide were added to a 0.05 M Tris hydrochloride buffer solution (pH 8.0) to give concentrations of 18.1 %, 10 mM, 0.15 m and 0.1 %, respectively, to prepare standard antigen diluents. Accordingly a set consisting of the foregoing solid phase first antibody, enzyme marker antibody, reaction diluents, substrate solution, reaction terminating solution and standard antigen diluents and lyophilized hydroneritoneum of a hecatom2 patient which was employed as a standard antigen was obtained as the reagent of the present invention. Example 2 M.onoclonal antibody 5C2 was dissolved in a 0.05 phosphate bu:LfeL solutinn (pH 7.5) to give a protein concentration of 1 mg/mS. To 10 # of the obtained solution, 50 # of 1 mCi of 125T-Na and 10 # of a solution prepared by dissolving chlor2mine T in said phos phase buzzer solution to give a concentration of 1.5 mg/m# were added successivelv and the reaction mixture was stirred for 30 sec. Subsequently 100 # of a solution prepared by dissolving sodium metabisulfite in said phosphate buffer solution to give a concentration of 2 mg/mS was added to stop the reaction. To the reaction liquor, 100 # of a solution prepared by dissolving potassium iodide in said phosphate buffer solution to give a concentration of 10 mgXmS was added and the reaction mixture was immediately subjected to gel-*iltration with Sephadex G-50 to thereby separate the substance labelled with 125I from 125I. Speciffic radioactivity of the substance labelled with 125I thus obtained was approximately 5 to 20 Ci/ g. The second antibody labelled with 125I thus obtained was combined with the solid phase first antibody prepared in the same manner as described in Example 1 to give the reagent of the present invention zor radioimmunoassay.

Claims: Claims: 1. A method for assaying basic fetoprotein by the sandwich method using two an ibodies, said two antibodies being selected from monoclonal antibasic fetoprotein antibodies which each r,sact with antigenic determinants of the basic fetoprotein different from each other. 2. A reagent for assaying basic fetoprotein by the sandwich method using two antibodies, said two antibodies being selected from monoclonal antibasic fetoprotein antibodies which each react with antigenic determinants of the basic fetoprotein different from each other. Claims for Austria , 1. A method for assaying basic fetcprotein by the sandwich method using two antibodies, said two antibodies being selected from monoclonal antibasic fetoprotein antibodies which each react with antigenic determinants of the basic fetoprotein different from each other. 2. A method for preparing a reagent for assaying basic fetoprotein by the sandwich method using two antibodies, said two antibodies being selected from monoclonal antibasic fetoprotein antibodies which each react with antigenic determinants of the basic fetoprotein different from each other.

Inventors: HATTORI, NOBU, ISHII, MASARU, SHIMOZURU, YASUNORI, TANIGUCHI, MASARU, WATANABE, KEISUKE
Applicants: NIPPON KAYAKU KABUSHIKI KAISHA
Application year: 1984
Main classification: G01N33
IPC codes: G01N33