GB2222591A - Monoclonal antibodies - Google Patents

Abstract

Monoclonal antibodies are produced by immunizing an animal with an antigen derived from human undifferentiated cells, additionally immunizing it with an antigen derived from tumor cells, separating antibody-producing cells from the animal, fusing with myeloma cells to prepare hybridomas, and selecting and cultivating a hybridoma which produces the desired monoclonal antibody. A monoclonal antibody so produced (a) reacts with a sialic acid-containing glycoprotein of molecular weight about 600 KD, (b) does not substantially react with human liver, bladder, nerve or large intestine carcinoma cells, germ cell neoplasm cells, culture supernatants of these cells, and nonionic surfactant-solubilized components of ruptured products of these cells, (c) does not substantially read with normal human kidney, gall-bladder, leucocyte, placenta, spleen, muscular, liver, colon, intestinal, pancreatic, esophagal, brain or bone cells, and nonionic surfactant-solubilized components of ruptured products of these cells, (d) does not substantially react with normal human serum, and (e) has such reactivity with an antigen derived from human undifferentiated cells that the absorbance measured by a solid-phase EIA method is about 2 to about 5 times that measured on only the antigen derived from human undifferentiated cells by the same method. A further monoclonal antibody so produced reacts with an antigen composed of alpha -fetoprotein.

Description

MONOCLONAL ANTIBODIES AND PROCESS FOR PRODUCTION OF MONOCLONAL ANTIBODIES This invention relates to novel monoclonal antibodies which selectively react with certain tumorassociated antigens, and also to a novel process for production of monoclonal antibodies which selectively react with tumor-associated antigens.

Monoclonal antibodies which specifically recognize tumor-associated antigens are used as diagnostic reagents for tumors or in the missile therapy of tumors by utilizing an antigen-antibody reaction involving the tumor-associated antigens.

In the prior art (for example, U.S. Patent 4,609,548), these monoclonal antibodies are obtained by immunizing animals with the tumor-associated antigens to obtain antibody producing cells, fusing these cells with myeloma cells to prepare hybridomas (fused cells), and cultivating the hybridomas.

When the tumor-associated antigen (tumor marker) specific for the target tumor is an identified antigen, the antigen can be used to immunize the animals after purification and is effective for obtaining an antibody which specifically recognizes this antigen. If, however, the tumor-associated antigen is not identified, this process can hardly be applied. Specifically, when the tumor-associated antigen is not identified, the antigen cannot be purified, and there is no option but to use an antigen obtained by homogenizing the tumor cells.

This antigen contains other antigens, and an antibody specifically recognizing the tumor-associated antigen is very rarely produced selectively.

Accordingly, an antibody having specificity for the tumor-associated antigen is now obtained with a very low probability by immunizing an animal with the antigen derived from tumor cells.

For example, European Patent Application Publication No. 145949-A2 describes monoclonal antibodies which specifically react with antigens relating to tumors such as ovarian carcinoma, renal carcinoma, endometrial carcinoma, colon carcinoma, breast carcinoma and cervical carcinoma, which are obtained by a method similar to that described in the above- cited U. S. Patent 4,609,548.

These publications, however, do not at all describe a monoclonal antibody which reacts specifically with ovarian carcinoma and lung carcinoma, it is hardly described elasewhere. The above-cited European Patent Application Publication No. 145949-A2 shows a monoclonal antibody "MH94" which is reactive with ovarian carcinoma and lumg carcinoma. This monoclonal antibody also shows reactivity with normal tissue cells.

The present inventors made extensive investigations in order to obtain an antibody specifically recognizing a tumor-associated antigen which is not identified, and consequently found a novel process for efficiently producing a monoclonal antibody which can recognize an unidentified, tumor-associated antigen. By this new process, the present inventors have succeeded in producing a monoclonal antibody which can recognize unidentified antigens associated with human ovarian carcinoma and human lung carcinoma, and also human liver carcinoma-related antigen.

According to one aspect of this invention, there is provided a process for producing a monoclonal antibody, which comprises immunizing an animal with an antigen derived from human undifferentiated cells, additionally immunizing it with an antigen derived from tumor cells, separating antibody-producing cells from the animal, fusing the antibody-producing cells with myeloma cells to prepare hybridomas, selecting from the hybridomas that hybridoma which produces the desired monoclonal antibody, cultivating the selected hybridoma, and recovering the monoclonal antibody secreted by the hybridoma.

The antigen derived from human undifferentiated cells ("HU antigen" hereinafter) is used in the first immunization in the process of this invention. The human undifferentiated cells, also known as human totipotency cells, are not completely differentiated into human organs or cells having a function, but have the ability to be differentiated into organs or cells having a function. Specifically, these cells are not differentiated into organs such as heart, liver and brain or blood cells such as lymphocytes.

The human undifferentiated cells exist, for example in 7 to 15-week old embryo human ("EHu" hereinafter), human amniotic fluids in the early stage of pregnancy and human bone marrow, most abundantly in 7-15, preferably 7-10, week old EHu. When the EHu exceeds 15 weeks, the human undifferentiated cells are differentiated into the organs and become antigenetically the same as adult cells. Hence, these differentiated cells cannot be used for the purpose of this invention. EHu can be obtained lawfully in accordance with the rules stipulated by Japan Society of Obstetrics and Gynecology.

The method of preparing the HU antigen is not particularly limited. A typical example of the method of preparing HU antigen from EHu is shown below.

It is possible to homogenize part or the whole of the tissues of EHu and use the supernatant as an antigen. But to decrease the selectivity of antibodyproducing cells produced by immunization with the antigen, it is preferable to remove differentiated organs such as small intestines and liver from the EHu, homogenize the remainder, and use the resulting supernatant as the antigen. Preferably, to use not only the surface antigen on the EHu cells but also the components within the cells as the antigen, various surface-active agents for rupturing the cell membranes are added to solubilize the cells. As a result of solubilization of the cells, all components of the undifferentiated cells can be utilized as the antigen. For solubilization of the cells, surface-active agents used conventionally for solubilization of cells may be used in this invention.

Specific examples of the surface-active agents include nonionic surface-active agents such as polyoxyethylene dodecyl ether, polyoxyethylene 2-methyl dodecyl ether, polyoxyethylene heptamethylhexyl ether, polyoxyethylene l-octylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene fatty acid esters and polyoxyethylene sorbitol ester; anionic surface-active agents such as cetyl trimethyl ammonium bromide, tetradecyl ammonium bromide and dodecyl pyridinium chloride; cationic surface-active agents such as sodium dodecylsulfate, sodium dodecylsulfonate and sodium dodecyl-N-sarcosinate; and amphoteric surface-active agents such as palmitoyl lysolecithin, dodecyl-N-betaine and dodecyl-beta-alanine.

A crude undifferentiated cell solubilized product obtained by solubilizing the human undifferentiated cells by the above method may be used directly as the HU antigen, but preferably it is centrifuged to remove insoluble components prior to use as the antigen.

If the insoluble components are mixed, the antigen has the low ability to produce an antibody, and is undesirable for immunization.

Animals generally used for immunization can be used in this invention. Specific examples include mammals such as mouse, rat, rabbit, goat, sheep, bovine and horse. The mouse and rat are preferred in view of the ease of obtaining myeloma cells to be fused with antibody-producing cells (B cells) obtained by immunization. Strains of the mouse and rat are not particularly limited, and may include mouse strains A, AKR, BALB/c, BDP, CBA, CE, C3H, C57BL, C57BR, C57L, DBA, FL, HTH, HTI, LP, NZB, NZW, RF, Rill, SJL, SWR, WB and 129, and rat srains Lou, Lewis, Spraque Dawley, ACI, BN and Fischer.In view of the compatibility of fusion with myeloma cells to be described mice of BALB/c strain and rats of Lou strain are most preferred animals to be immunized. The ages of the mice and rats are preferably 5 to 12 weeks, more preferably 6 to 8 weeks. If they are less than 5 weeks old, immunization is difficult. If they are more than 12 weeks old, the immunization efficiency tends to decrease. The immunization of an animal with the HU antigen in this invention may be carried out by a known immunizing method, for example the methods described in detail in, for example, D. M. Weir: Handbook of Experimental Immunology, Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978) and E. A. Kabat and M. M. Mayer: Experimental Immunochemistry, Charles C. Thomas Publisher, Springfield, Illinois (1964).

Immunizing methods that can be suitably used in this invention are specifically shown below. The HU antigen may be administered intraperitoneally or through the vein. To increase the immunizing efficiency, the antigen is preferably administered both intraperitoneally and through the vein. To increase the immunizing efficiency particularly, it is preferred to administer it intraperitoneally in the first half of the immunization procedure, and through the vein in the latter half or only in the final immunization. The immunization schedule cannot be generalized because of the types and the individual differences of the animal to be immunized.

Generally, the number of administrations of the HU antigen is 3 to 6, preferably 3 to 4, and the interval of administrations is 2 to 6 weeks, preferably 3 to 4 weeks.

If the number of administrations is increased excessively, the valuable HU antigen is wasted, and if the administration interval is broadened, the activity of the cells will be undesirably lowered. The amount of HU antigen used to immunize cannot be generically determined because it will vary depending upon the types of the animal to be immunized, the individual differences, etc.

Suitably, it is generally 0.05 to 5 ml, preferably 0.1 to 0.5 ml.

The important requirement in the process of this invention is that after the above immunization with the HU antigen, the animals is additionally immunized with an antigen derived from tumor cells ('TC antigen" hereinafter). As a result of immunization with the HU antigen, antibody-producing cells of the kind corresponding to the HU antigen are formed within the immunized animals. By additionally immunizing it with a specific TC antigen, clones of antibody-producing cells which specifically recognize the TC angigen can be increased selectively from the antibody-producing cells. Accordingly, the efficiency of fusion between the antibodyproducing cells with myeloma cells in the cell fusion to be described hereinafter can be increased.Consequently, by cultivating a hybridoma obtained by cell fusion, the desired monoclonal antibody can be produced with good efficiency.

The previously known method of additional immunization comprises immunizing an animal with a TC antigen and then further with the same kind of TC antigen. By this method, antibody-producing cells to be increased by additional immunization are limited to those which are produced in a relatively high proportion in the previous immunization. Accordingly, if an antibody produced by the antibody-producing cells does not specifically recognize a tumor-associated antigen in the TC antigen, the additional immunization becomes quite insignificant.

The TC antigen used in the additional immunization in this invention may be properly selected from TC antigens of a tumor corresponding to the desired monoclonal antibody. For example, when it is desired to obtain a monoclonal antibody showing specific reactivity with human ovarian carcinoma, the TC antigen of human ovarian carcinoma is used. The TC antigen may be prepared by using available and known carcinoma cells such as human stomach carcinoma, human liver carcinoma, human lung carcinoma, human bladder carcinoma, human pancreatic carcinoma, humman renal carcinoma and human large intestine carcinoma according to the desired monoclonal antibody. In the preparation of the TC antigen from the tumor cells, the carcinoma cells may be ruptured and directly used.Preferably, the carcinoma cells are solubilized by using the same surface-active agents as described above, and used as an additional immunization antigen after they are centrifuged to remove insoluble materials.

The additional immunization may be carried out intraperitoneally or through the vein. To increase the efficiency of the additional immuinization, the latter route is preferred.

Preferably, the additional immunization is carried out after the lapse of 1 to 6 weeks, preferably 2 to 4 weeks, more preferably 2 to 3 weeks, after the final immunization of the animals with the HU antigen. Usually, the additional immunization is carried out once, and preferably spleen cells containing antibody-producing cells are taken out from the immunized animals after the lapse of 1 to 10 days, preferably 2 to 5 days, more preferably 2 to 3 days, from the additional immunization.

The effect of the additional immunization is little if it is carried out later than 6 weeks or earlier than 1 week from the previous immunization. Furthermore, if the time of extracting the spleen cells is after more than 10 days from the additional immunization, cells are prone to form which produce an antibody to the TC antigen additionally used in the immunization. If it is before 1 day from the additional immunization, the effect of the additional immunization tends to be small.

The suitable amount of the TC antigen used at the time of the additional immunization differs with the kind and size of the immunized animal, and cannot be generally determined. For mice, it is generally 0.05 to 5 ml, preferably 0.1 to 0.5 ml, more preferaly 0.1 to 0.2 ml. Administration of an unnecessarily large amount of the antigen decreases the immunizing efficiency, and is not desirable for the animal immunized.

For the separation of antibody-producing cells from the spleen cells taken out aseptically from the immunized animal may be carried out by known methods, for example, the methods described in Koehler et al., Nature, 256, 495 (1975), Koehler at al., Eur. J. Immunol. 6, 511 (1977), Milstein et al., Nature 266, 550 (1977) and Walsh, Nature, 266, 495 (1977). For example, a general method may comprise mincing the cells, filtering the minced product on a stainless mesh, floating the filtrate on Eagle's minimum essential medium (MEM), and separating the antibody-producing cells.

In order to obtain a monoclonal antibody, the antibody-producing cells are fused with myeloma cells to obtain hybridomas.

The myeloma cells used in the cell fusion is not particularly limited. They may be selected from myeloma cell lines used frequently in cell fusion, for example, myeloma cells derived from mice and myeloma cells derived from humans. Specific examples include X63-Ag8(X63), NSI-Ag4/1(NSI), P3X63-Ag8Ul(P3Ul), X63-Ag8.653(X63.653), SP2/0-Agl4(SP2/0), MPC11-45.6TG1.7(45.6TG), FO, S149/5XXO and BU.1 derived from mice; and U-2666AR(SKO-007), GM1500.6TG-A12(GM1500), UC729-6, LICR-LON-AMy2(HMy2), and 8226AR/NIP4-1 (NP41) derived from humans (the parenthesized designations are abbreviations).Preferably, the myeloma cell line is an HGPRT line (hypoxanthine-guanine phosphoribosyl-transferase)deficient cell line for which a technique of selecting a hybridoma after the cell fusion has already been established. The above-exemplified cell lines are all HGPRT-deficient.

The fusion of the antibody-producing cells with the myeloma cells may be carried out by a known method such as a chemical method involving mixing the antibodyproducing cells with the myeloma cells in a high concentration solution of a polymer such as polyethylene glycols, or a physical method utilizing an electrical stimulation. It can be carried out under conditions which do not drastically decrease the survival rate of the cells isee, for example, D. M. Weir: Handbook of Experimental Immunology, Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978) and E. A. Kabat and M. M. Mayer: Experimental Immunochemistry, Charles C.

Thomas Publisher, Springfield, Illinois (1964)1. For example, the above chemical method is generally practiced by the following procedure. A high concentration solution of polyethylene glycol having a molecular weight of 1,500 to 6,000, preferably 2,000 to 4,000, is used, and at a temperature of 30 to 40 C, preferably 35 to 38 C, the antibody-producing cells are mixed with the myeloma cells for 1 to 10 minutes, preferably 5 to 8 minutes.

There is no particular restriction on the method of selecting hybridomas obtained by the cell fusion. Usually, the HAT (hypoxanthine-aminoputerinthymidine) selection mehtod is employed. The details of the HAT selection method are described in Koehler et al.: Nature, 256, 485 (1975) and Milstein et al.: Nature, 266, 550 (1977). This method is effective for obtaining hybridomas by using HGPRT-deficient myeloma cells which cannot survive in aminoputerin. Specifically, by cultivating the hybridomas obtained by the cell fusion in HAT medium (medium containing hypoxanthine, aminoputerin and thymidine, those hybridomas which also have resistance to aminoputerin are selectively left in the medium and proliferated.Cloning of the hybridomas can be carried out by known methods such as the methyl cellulose method, the soft agarose method and the limiting dilution method (see, for example, B. M. Barbara and M. S. Stanley: Selected Methods in Cellular Immunology, W. H. Freeman and Company, San Francisco (1980)1, The limiting dilution method is especially suitable. According to this method, a feeder such as a fibroblast cell line derived from a rat embryo, normal mouse spleen cells, thymus cells or ascites cells is inoculated in a microplate. On the other hand, the hybridomas are diluted in a medium to a concentration of 0.2 to 0.5 cell/0.2 ml, and the diluted hybridoma suspension is put in an amount of 0.1 ml per well.Every predetermined period, for example every 3 days, about 1/3 of the medium is replaced with a fresh one. When the cultivation is continued for about 2 weeks, clones of the hybridomas are proliferated. By cultivating the hybridomas so selected, a monoclonal antibody can be produced with good efficiency. Preferably, prior to the cultivation, the hybridomas are screened to choose hybridomas which produce the desired monoclonal antibody. The screening may be performed by known methods such as solid-phase EIA (enzyme immunoassay), liquid-phase EIA, solid phase RIA (radioimmunoassay), liquid-phase RIA, and a fluorescent antibody technique. Since in this invention, there is a possibility that an antibody to quite an unknown antigen will be formed, it is preferable to use the solid-phase EIA method.By this method, the tumor cell-associated antigen is fixed to each of the wells of the microplate, and then the supernatant containing the hybridoma is added to induce an antigen-antibody reaction. The wells are then washed, and a labelled antibody such as a peroxidase-labelled anti-mouse IgG antibody or a peroxidase-labelled anti-mouse IgM antibody is added. The wells were further washed, and hydrogen peroxide as a substrate and a coloring agent are added. The absorbance is then measured, and the activity is also measured. By this method, hybridomas producing the desired monoclonal antibody can be obtained. This screening may be carried out before or after the cloning of the hybridomas as above.

In the present invention, the method of cultivating the hybridomas is not particularly limited, and may be the same as the ordinary method of cultivating hybridomas. For example, the hybridomas may be cultivated in the same medium as used in the above cloning procedure. To obtain the monoclonal antibody in great quantities, it is possible to inject the hybridomas into the abdominal cavity of mice, and extract the monoclonal antibody from the ascites. In this method, an immunosuppressant is intraperitoneally injected into mice of the same strain as the hybridoma to inactivate T cells.

Then, 7 to 107 clone cells are suspended in a serum-free medium, and introduced intraperitoneally.

Usually, in 10 to 20 days when the abdomen swell, the ascites is taken from the mice. This method enables the monoclonal antibody to be obtained in a concentration of at least 100 times as large as that obtained from the culture.

There is no particular restriction on the method of purifying the monoclonal antibody obtained by this method. For example, the purifying methods described in Weir: Handbook of Experimental Immunology, Vol. I, II, III cited above may be used. Typical methods include ammonium sulfate salting out, gel filtration, ion-exchange chromatography and affinity chromatography.

The monoclonal antibody could be purified by repeating salting out with ammonium sulfate 3 to 4 times, preferably 3 to 6 times. However, with this method, the yield of the purified monoclonal antibody is very low.

The monoclonal antibody can be purified to a high purity by subjecting it to salting out with ammonium sulfate once or twice, and purify it further by one or more, preferably two, methods selected from gel filtration, ion exchange chromatography and affinity chromatography. The combination of the ammonium sulfate salting out with the other methods may be, for example, (1) ammonum sulfate salting out - ion exchange chromatography - gel filtration, (2) ammonuim sulfate salting out - ion exchange chromatography - affinity chromatography, and (3) ammonium sulfate salting out - gel filtration - affinity chromatography, each in the order stated.

The combination (3) is most suitable to obtain a monoclonal antibody of high purity in a high yield.

The hybridoma producing the above monoclonal antibody can be stored in the frozen state in liquid nitrogen or in a refrigerator at -80 0C or below.

Specific examples of the hybridomas so produced include hybridoma 5Fll and hybridoma 6H13 deposited at Fermentation Research Institute, Agency of Industrial Science and Technology, Tsukuba, Ibaraki-ken, Japan under FERM BP-1997 and FERM BP-2525 under the Budapest Treaty.

Examples of the novel monoclonal antibodies produced by the process of this invention described above are (i) a monoclonal antibody (I) produced by a hybridoma (for example, hybridoma SF11 (FERM BP-1997)1 which is prepared by fusion of antibody-producing cells from a mouse immunized with an antigen derived from human undifferentiated cells (HU antigen) and additionally with a TC antigen derived from human ovarian carcinoma cells with mouse myeloma cells; and (ii) a monoclonal antibody (II) produced by a hybridoma (for example, hybridoma 6H13 (FERM BP-2525)1 which is prepared by fusion of antibody producing cells from a mouse immunized with an antigen derived from human undifferentiated cells (HU antigen) and additionally with a TC antigen derived from human liver carcinoma cells, with mouse myeloma cells.

The characteristics of these monoclonal antibodies (I) and (II) will be described below in detail.

Monoclonal antibody (I) The monoclonal antibody (I) shows the following reaction characteristics.

(a) It reacts with an antigen composed of a sialic acid-containing protein having a molecular weight of about 600 KD.

(b) It does not substantially react with human liver carcinoma cells, human bladder carcinoma cells, human nerve carcinoma cells, human large intestine carcinoma cells, human embryonic carcinoma cells, culture supernatants of these cells, and nonionic surfactantsolubilized components of ruptured products of these cells.

(c) It does not substantially react with normal human kidney cells, normal human gallbladder cells, normal human leucocyte cells, normal human placenta cells, normal human spleen cells, normal human muscular cells, normal human liver cells, normal human colon cells, normal human intestinal cells, normal human pancreatic cells, normal human esophageal cells, normal human brain cells, normal human bone cells, and nonionic surfactant-solubilized components of ruptured products of these cells.

(d) It does not substantially react with normal human serum.

(e) Its reactivity with an antigen derived from human undifferentiated cells is such that the absorbance measured by a solid-phase EIA method is about 2 to about 5 times that measured on only the antigen derived from human undifferentiated cells by the same method.

The monoclonal antibody (I) provided by the present invention strongly reacts with an antigen composed of a sialic acid-containing glycoprotein having a molecular weight of about 600 KD (kilodaltons), and therefore can be used to identify and determine substances containing the sialic acid-containing glycoprotein as a component. The sialic acid-containing glycoprotein is a protein containing sialic acid in the sialic acid chain portion. When this protein is treated with 40 % neuraminidase solution, its reactivity with the monoclonal antibody is lost, and when it is treated with trypsin in a high concentration, its reactivity with the monoclonal antibody remains. It is presumed therefore that this monoclonal antibody (I) recognizes the bonded portion of the sialic acid chain and the peptide chain of this sialic acid-containing glycoprotein.

The molecular weight of the sialic acidcontaining glycoprotein is measured by SDS-PAGE electrophoresis and immunoblotting. The sialic-acid containing glycoprotein exists within human ovarian carcinoma cells and human lung carcinoma cells, and/or is released into the blood from these cells. It has been determined that the monoclonal antibody (I) of this invention strongly reacts with human epithelial ovarian carcinoma cells, human lung carcinoma cells, culture supernatants of these cells, nonionic surfactant-solubilized components of these cells, the serum of a patient with epithelial ovarian carcinoma, and the serum of a patient with lung carcinoma.

It has also been determined that the monoclonal antibody (I) of this invention does not substantially react with tumor-associated antigens which do not contain, nor release, the sialic acid-containing glycoprotein, such as human liver carcinoma cells, human bladder carcinoma cells, human nerve carcinoma cells, human large intestine carcinoma cells, human embryonic carcinoma cells, culture supernatants of these cells, and nonionic surfactant-solubilized components of ruptured products of these cells.

It has also been determined that the monoclonal antibody (I) of this invention does not substantially react with most human normal cells, for example normal human kidney cells, normal human gallbladder cells, normal human leucocyte cells, normal human placenta cells, normal human spleen cells, normal human muscular cells, normal human liver cells, normal human colon cells, normal human intestinal cells, normal human pancreatic cells, normal human esophageal cells, normal human brain cells, normal human bone cells, nonionic surfactant-solubilized components of ruptured products of these cells, and also with normal human sera. However, the monoclonal antibody (I) weakly react with the antigen (HU antigen) derived from human undifferentiated cells used as the immunogen.Specifically, its reactivity with an antigen derived from human undifferentiated cells is such that the absorbance measured by a solid-phase EIA method is about 2 to about 5 times that measured on only the antigen derived from human undifferentiated cells by the same method.

Furthermore, the monoclonal antibody (I) weakly reacts with normal human lung cells, normal human ovarian cells and normal human thyroid gland. Specifically, its reactivity with nonionic surfactant-solubilized components of ruptured products of normal human lung cells, normal human ovarian cells and normal thyroid gland cells is such that the absorbance measured by a solid-phase EIA method is about 2 to about 5 times that measured on the above solubilized components alone measured by the same method.

This reactivity is due presumably to cross reaction.

However, the above weak reactivity of the monoclonal antibody (I) of this invention with the HU antigen and normal human lung cells, normal human ovarian cells and normal human thyroid gland cells is much weaker than its reactivity with the sialic acid-containing glycoprotein, and does not constitute a substantial obstacle in detecting the sialic acid-containing glycoprotein using the monoclonal antibody (I).

In the present invention, the reactivity of the monoclonal antibodies with various antigens is determined by solid-phase enzyme immunoassay (see, for example, D. M. Weir: Handbook of Experimental Immunology, Vol.

I, II, III, Blackwell Scientific Publications, Oxford (1978)1 and the ABC method on formalin-fixed paraffin slices [Avidin Biotinylated Horseradish Peroxidase Complex assay: Hsu et al.: J. Hostochem. Cytochem., 29, 577 (1981)J.

The expression that an antibody ;does not substantially react" with regard to the reactivity of the monoclonal antibody with the nonionic surfactantsolubilized components of the ruptured products of the subject cells means that the absorbance measured by the solid-phase EIA is less than 5 times that (blank value) measured on the solubilized components alone by the same method. The expression that it "reacts strongly" means that the absorbance measured by the above method is more than 30 times the blank value.

The monoclonal antibody (I) of this invention relates to a class of IgM.

A suitable example of the HU antigen used in the production of the monoclonal antibody (I) is a solubilized component obtained by treating a ruptured product of cells of a human 7 to 10 week old-embryo with the aforementioned nonionic surfactants. A suitable example of the antigen derived from human ovarian carcinoma cells is a solubilized component obtained by treating a ruptured product of human ovarian carcinoma cells with the aforementioned nonionic surfactants.

Monoclonal antibody (II) The monoclonal antibody (II) shows the following reaction characteristics.

(a) It reacts with an antigen composed of O(-fetoprotein.

(b) It does not substantially react with human ovarian carcinoma cells, human lung carcinoma cells, human bladder carcinoma cells, human nerve carcinoma cells, human large intestine carcinoma cells, human embryonic carcinoma cells, culture supernatants of these cells, and nonionic surfactant-solubilized components of ruptured products of these cells.

(c) It does not substantially react with normal human kidney cells, normal human thyroid gland cells, normal human gallbladder cells, normal human leucocyte cells, normal human placenta cells, normal human spleen cells, normal human muscular cells, normal human lung cells, normal human liver cells, normal human colon cells, normal human intestinal cells, normal human pancreatic cells, normal human esophageal cells, normal human brain cell, normal human bone cells, and nonionic surfactant-solubilized components of ruptured products of these cells.

(d) It does not substantially react with normal human serum.

(e) Its reactivity with an antigen derived from human undifferentiated cells is such that the absorbance measured by a solid-phase EIA method is about 2 to about 5 times that measured on only the antigen derived from human undifferentiated cells by the same method.

The monoclonal antibody (II) provided by this invention strongly reacts with an antigen composed of & fetoprotein, and therefore can be used to identify and determine substances containing & fetoprotein as a component. When an authentic sample of i-fetoprotein is treated with a 40 % neuraminidase solution, its reactivity with the monoclonal antibody (I) is lost. In contrast, when it is treated with trypsin, its reactivity is not lost even if the trypsin is high in concentration.

Accordingly, the monoclonal antibody (II) appears to recognize the sialic acid chain portion of 4-fetoprotein.

It is known that i-fetoprotein exists within human liver carcinoma cells, and/or is released into the blood from the cells Esee T. M. Chu: sAlpha-fetoprotein" in Biochemical Markers for Cancer, Maareel Dekker, New York (1982)1. It has been determined that the monoclonal antibody (II) strongly react with human liver carcinoma cells, a culture supernatant of human liver carcinoma cells, and a nonionic surfactant-solubilized component of a ruptured product of liver carcinoma cells, and also with the serum of a patient with liver carcinoma.

It has been determined on the other hand that the monoclonal antibody (II) does not substantially react with tumor-associated antigens which do not contain d-fetoprotein nor release it, for example, human ovarian carcinoma cells, human nerve carcinoma cells, human large intestine carcinoma cells, human embryonic carcinoma cell, culture supernatants of these cells, and nonionic surfactant-solubilized components of ruptured products of these cells.

It has also bern determined that the monoclonal antibody (II) of this invention does not substantially react with most human normal cells, for example normal human kidney cells, normal human gallbladder cells, normal human leucocyte cells, normal human placenta cells, normal human spleen cells, normal human muscular cells, normal human liver cells, normal human colon cells, normal human intestinal cells, normal human pancreatic cells, normal human esophageal cells, normal human brain cells, normal human bone cells, nonionic surfactant-solubilized components of ruptured products of these cells, and also with normal human serum. However, the monoclonal antibody (II) weakly react with the antigen (HU antigen) derived from human undifferentiated cells used as the immunogen.Specifically, its reactivity with an antigen derived from human undifferentiated cells is such that the absorbance measured by a solid-phase EIA method is about 2 to about 5 times that measured on only the antigen derived from human undifferentiated cells by the same method.

Furthermore, because of a cross reaction on the basis of cell similarity, the monoclonal antibody (II) weakly reacts with an extract of normal human liver cells. However, the above weak reactivity of the monoclonal antibody (II) with the HU antigen and normal human liver cells is much weaker than that with i-fetoprotein, and does not cause a substantial trouble in the detection of d-fetoprotein utilizing the monoclonal antibody (it).

The reactivity of the monoclonal antibody (II) with nonionic surfactant-solubilized component of a ruptured product of normal human liver cells is such that the absorbance measured by the solid-phase EIA is about 2 to about 5 times that measured by the same method on the solubilized components alone.

A suitable example of the HU antigen used in the production of the monoclonal antibody (I) are a solubilized component obtained by treating a ruptured product of cells of a human 7 to 10 week old-embryo with the aforementioned nonionic surfactants. A suitable example of the antigen derived from human liver carcinoma cells is a solubilized component obtained by treating a ruptured product of human liver carcinoma cells with the aforementioned nonionic surfactants. The monoclonal antibodies (I) and (II) have the property of being able to specifically recognizing a sialic acid-containing glycoprotein and i-fetoprotein, respectively.By utilizing this property, the monoclonal antibody (I) can be used to diagnose ovarian carcinoma and lung carcinoma with a very high accuracy by measuring the concentration of antigens in body fluids by the radio immunoassay described in D. M. Weir: Handbook of Experimental Immunology, Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978) or solid-phase EIA or a fluorescent antibody technique, or by imaging the radioactivity of the monoclonal antibody (I) labelled with radioisotopes. It can also be applied to immunotherapy using ovarian carcinoma or lung carcinoma tissues used as a target, or to missile therapy with an anticancer agentmonoclonal antibody complex which specifically carries the anticancer agent to ovarian carcinoma or lung carcinoma.The monoclonal antibody (II) can be applied to the diagnosis and treatment of liver carcinoma by a similar method.

The following Examples illustrate the present invention more specifically.

EXAMPLE 1 1) Preparation of a human undifferentiated cell antigen (HU antigen) A dead embryo, 7 to 10 weeks of age, was obtained lawfully in accordance with the rules stipulated by Japan Society of Obstetrics and Gynecology. The differentiated liver and intestines were removed from the embryo, and 150 ml of 0.lM phosphate buffer (pH 7.4) containing 0.1 % NP-40 was added to the treated embryo.

The mixture was homogenized, and the homogenate was stirred overnight at 4 C to rupture, and the insoluble components were separated by centrifugation (10,000 rpm, 1 hour). The supernatant was passed through a Millipore filter (0.4in; a product of Japan Millipore Co.), and used as an antigen. The protein concentration of the antigen was 10 mg/ml.

2) Preparation of an antigen derived from ovarian carcinoma (TC antigen) for additional immuniza tion Human ovarian carcinoma tissues were ruptured in 0.1M PBS containing 0.1 % NP-40 and stirred overnight at 4 C to solubilize and extract them. The extract was centrifuged to remove the insoluble components. The resulting extract was passed through a Millipore filter (0.45yam; a product of Japan Millipore Co.), and diluted with 0.lM PBS so that its protein concentration became 5 mg/ml.

3) Immunization of BALB/c mice The antigen obtained in 1) above was intraperitoneally administered to BALB/c strain mice three times in an amount of 0.1 ml each time at intervals of 2 weeks. As a final administration, 0.1 ml of the antigen obtained in 1) was administered to the same mice through the vein. Two weeks after the final administration, 0.1 ml of the QC antigen prepared in 2) was administered to the same mice through the vein to immunize the mice additionally.

4) Cell fusion Three days after the additional immunization, spleen cells were extracted from the immunized mice. The spleen cells were minced, compressed and filtered by a stainless mesh, and suspended in Eagle's MEM to obtain a spleen cell suspension. The spleen cell suspension and mouse myeloma cells NS-1 were each washed three times with serum-free MEM, and mixed at a ratio of 10:1. The mixture was centrifuged (800 rpm, 5 minutes). The precipitate was disintegrated, and 1 ml of 44 % polyethylene glycol 2000/MEM solution was gradually added. The mixture was incubated at 37 C for 8 minutes to perform cell fusion. Eight minutes later, 1 ml of MEM was added.

Furthermore, MEM was added at a rate of 2 ml/min. After its total amount reached 10 ml, the mixture was centri fuged at 1000 rpm for 5 minutes. The supernatant was removed. The resulting cell precipitate was suspended in RPMI 650 medium containing 10 % bovine fetal serum so that the concentration of NS-1 became 1 x 104 cells/ml.

The suspension was inoculated in a 96-well microplate at a rate of 0.1 ml per well. One day later, RPMI 1640 10 % FCS medium containing HAT (hypoxanthine 1 x 10 4 aminoputerin 4 x 10-7 M, thymidine 1.6 x 10-5 M) (to be referred to as HAT -mediumj was added at a rate of 0.1 ml per well. Thereafter, half of the medium was replaced by HAT medium every 3 to 4 days to select hybridomas using HAT medium. In 10 to 14 days, the hybridoma proliferated in almost all wells. At this time, the occurrence ratio of hybridomas was 18.7 %.

5) Selection of antibody-producing cells The following TC angiten were provided.

SK-MES-1, ME 180 and MBI derived from human lung carcinoma; LI-7, HuH-7 and HC-4 derived from human liver carcinoma; HuB-4, HuB-15, HU-15N and HuB-40 derived from human bladder carcinoma; SR-N-FI, SK-N-AS and SK-N-DZ derived from human nerve carcinoma; CO-3 derived from human large intestine carcinoma; NEC derived from human ovarian carcinoma. Furthermoe, the following normal human cells were provided. Human kidney cells, human thyroid gland cells, human gallbladder cells, human leucocyte cells, human placenta cells, human spleen cells, human muscular cells, human lung celles, human liver cells, human colon cells, human intestinal cells, human pancreatic cells, human esophageal cells, human brain cells, human bone cells, and HU cells.

SK-MES-1 cells were ruptured in 0.1M PBS containing 0.1 % NP-40, and stirred overnight at 4 0C to solubilize and extract the cells. The extract was centrifuged and the insoluble components were removed. The extract was diluted with 0.lM PBS so that its protein concentration became 0.5 mg/ml. The diluted extract was added to a 96-well microplate for EIA at a rate of 50 A per well. The plate was then incubated at 37 C for 2 hours. The plate was washed three times with PBS containing 0.05 % Tween 80 (T-80PBS). 50fl1 of 0.1 % bovine serum albumin (BSA) was added to each well, and the plate was stored at 4 OC (tumor cell-fixed plate).By the same procedure, TC antigen-fixed plate and normal cell-fixed plates were prepared by using the above TC antigens and normal cells. The stored TC cell-fixed plate was washed three times with T-80PBS, and the culture supernatant obtained in 4) above was added at a rate of 50jc1 per well. The plate was incubated at 37 OC for 1 hour and then washed with T-80PBS three times. A horse radish peroxidase-labelled antimouse IgG antibody was diluted to 1000-fold and added to the microplate at a rate of 50 ss per well, followed by incubation at 37 C for 1 hour.

The plate was washed three times with T-80PBS, and a coloring solution containing 60 ml of 0.02M phosphatecitrate buffer (pH 8.0), 5 microliters of aqueous hydrogen peroxide and 50 mg of ABTS was added to the plate at a rate of 100 ss per well to induce coloration.

The reaction was then stopped by adding 0.25 % HF solution, and the absorbance of the solution at 510 nm was measured to determine the reactivity of the antibody produced from the antibody-producing cells with each of the antigens tested. The reaction conversion with the TC antigen was 15.3 %.

6) Cloning of hybridoma Hybridomas in wells which showed reactivity in 5) above were taken out, and diluted with RPMI 1640 medium containing 10 % bovine fetal serum. The mixture was inoculated in a microtray at a rate of 0.5 cell/well.

Mouse intraperitoneal cells as feeder cells had been inoculated in the microtray at a density of 2 x 106/ml and cultivated. While replacing the medium, the cultivation was continued for about 2 weeks. Antibodies in those wells in which colonies of the hybridomas appeared were measured by the method shown in 5) above, and those hybridomas which showed positiveness to the TC antigen were selected and again cloned. The hybridoma 5Fll obtained was deposited in Fermentation Research Institute, Tsukuba-shi, Ibaraki-ken, Japan under FERM BP-1997.

7) Preparation of a monoclonal antibody Pristan (a product of Aldrich Co.) was intraperitoneally administered to BALB/c strain mice, more than 7 weeks old, in an amount of 0.5 ml. More than one week later, proliferated hybridoma cells 5Fll was inoculated intraperitoneally at a rate of 1 to 9 x 105 cells/mouse. The mice inoculated with hybridoma 5Fll abruptly began to increase in body weight one week later, and the increase reached a peak in 10 to 15 days. The ascites was extracted from the mice when their body weight were approximately at their peak. The ascites was centrifuged at 3,000 rpm for 10 minutes to obtain 5 to 15 ml/mouse of ascites containing a monoclonal antibody.

8) Purification of the monoclonal antibody The monoclonal antibody was purified from 10 ml of the ascites obtained in 7) above in accordance with the method of Hudson et al. (Practical Immunology, Blackwell Sci. Pub., 1976). A saturated aqueous solution of ammonium sulfate (10 ml) was added to 10 ml of the ascites. The mixture was left to stand and centrifuged.

The resulting precipitate was dissolved in 5 ml of 0.1M phosphate buffer (PB), and the solution was dialyzed against 500 ml of 0.lM PB. After the dialysis, the dialyzate was centrifuged at 10,000 rpm for 10 minutes to obtain a supernatant. The supernatant was applied to a DEAE Sepharose column (a product of Pharmacia Co.). The column was washed with PB, and then sujected to linear gradient by salt concentrations to elute antibody fractions. The resulting antibody fractions were subjected further to Sephadex G-200 column (a product of Pharmacia Co.) to give 153 v of monoclonal antibody 5Fll.

9) Reactivity of the monoclonal antibody (a) Reactivity with cell extracts The monoclonal antibody obtained in 8) above was diluted with PB to a concentration of 10 > gg/ml. By using the resulting solution instead of the culture supernatant in 8), the same procedure was repeated on the TC antigen-fixed plates, the normal cell-fixed plates and HU antigen-fixed plate provided in 5). The reactivities of the monoclonal antibody with extracts of the TC antigens, the normal cells and the HU antigen were determined, and the results are shown in Table 1.

(b) Reactivity with cell culture supernatants The TC antigen cells indicated in 5) were subcultivated and conditioned in a serum-freee medium (GIT, Daigo T medium produced by Japan Pharmaceutical Co., Ltd.). The culture supernatants were each concentrated so that the protein concentration became 1 mg/ml, and by the same way as in 5). Plates to which TC antigen cell culture supernatants were fixed were obtained. The reactivities of the monoclonal antibody were determined by the same method as in 9), (a) on the TC antigen cell culture supernatant-fixed plates. The results are shown in Table 2.

(c) Reactivity with cells The reactivities of the monoclonal antibodies with the TC antigen cells and normal cells provided in 5) above were examined by using immuno staining tissue (Hsu et al.: J. Histochem. Cytochem., 29, 577-581, 1981) in accordance with the ABC method on paraffin slices. The paraffin slices were prepared by fixing cells with 10 % phosphate buffer/formal in, embedding them in paraffin, slicing the mass, and attaching the slices to slide glasses. Then, the paraffin was removed for 20 minutes and the slices were washed with PBS and then immersed in 0.25 % trypsin solution at 37 OC for 1 hour.After immersion, they were washed with PBS and furher immersed in 0.3 % hydrogen peroxide solution at room temperature for 30 minutes, followed by masking with normal goard serum (NGS), and reacted with the monoclonal antibody in a concentration of 1 mg/ml for 1 hour. After the reaction, the slices were washed with PBS. Biotin-labelled goat antimouse IgG (a product of Tago Co.) was diluted to 20-fold and reacted. The slices were washed, and then reacted with 0.5 ml of the ABC solution prepared from VECTASTAIN ABC kit. The slices were further washed with PBS. They were further reacted with 0.005 % H202diaminobenzidine tetrahydrochloride (DAB) solution. They were washed further with PBS, then stained with 1 % Methyl Green and sealed in glycerol gelatin. The sample after the sealing was observed under an optical microscope.From the degree of staining of the cells, the reactivity of the cells with the monoclonal antibody was examined. The results are shown in Table 3.

10) Ig class of the monoclonal antibody 1 % agarose was added to 1/15 M-PBS, and the mixture was boiled to form a solution. The solution was then solidified to a thickness of 1 mm on a slide glass.

Holes having a diameter of 3 mm were formed at 3 mm intervals. To each of the holes were added 15/ 1 of antimouse Ig class serum and 15/1 of a solution of the monoclonal antibody obtained in 8) above, and the slide glass was left to stand in a humid box for 10 hours. The monoclonal antibody obtained in 8) above showed a precipitation line based on an antigen-antibody reaction in the antimouse IgM serum.

11) Identification of an antigen recognized by the monoclonal antibody A 5 % acrylamide solution containing 0.1 % sodium dodecylsulfate (SDS) and 0.1 % sodium persulfate were injected between two glass plates (10 cm x 10 cm) in a slab electrophoresis device (made by ATTO Co,, Ltd.), and solidified to prepare two plates for SDS electrophoresis. 10yet1 of the TC antigen obtained in 2) was added to 40/1 of a 2mM sodium dodecylsulfate (SDS) solution containing 0.01 % Bromo Phenyl Blue (a product of Sigma Co.), and mixture was heated at 100 0C for 1 minute. After heating, the mixture was added to each of the SDS electrophoretic plates. A current was passed through the plates at 5 mA for 10 hours to perform electrophoresis.After the passing of current, one plate was stained with Coomassie Brilliant Blue (a product of Sigma Co.), and the molecular weight was measured. A nitrocellulose membrane was provided and the other plate was transferred to the nitrocellulose membrane by using a blotting device (made by ATTO Co., Ltd.). It was then reacted with the monoclonal antibody, and then with HRP-labelled antimouse IgM antibody. The nitrocellulose membrane was stained by using a coloring solution, and a band of an antigen was detected. At this time, the molecular weight of the antigen was about 600,000.

This antigen was treated with a 40 % neuraminidase solution at 37 C for 30 minutes, and in the same way as in 5), a tumor cell-fixed plate was prepared.

When the reaction of the antibody with the antigen was examined, the reactivity of the antibody disappeared.

This led to the presumption that the antigen contains a sialic acid chain, and this sialic acid chain is sialic acid.

EXAMPLE 2 HU antigen was prepared by the same method as in Example 1, 1). Then, a TC antigen was prepared from human liver carcinoma cells by the same method as in Example 1, 2). The mice was immunized and additionally immunized with these antigens in the same way as in Example 1, 3). After the additional immunization, cell fusion was carried out as in Example 1, 4), and antibodyproducing cells were selected from the hybridomas as in Example 1, 5). The fusion rate at this time was 18.6 %, and the reaction conversion was 15.3 %. Hybridoma 6H13 which was positive in reactivity was deposited in Fermentation Research Institute, Tsukuba-shi, Ibaraki-ken, Japan under FERM BP-2525.This hybridoma 6H13 was cloned by the same method as in Example 1, 6), and a monoclonal antibody was obtained and purified by the same method as in Example Is 7) and 8) from the hybridoma 6H13. The reactivity of the monoclonal antibody was measured as in Example 1, 9), and the results are shown in Tables 1 to 3.

EXAMPLE 3 HU antigen was prepared by the same method as in Example 1, 1). Then, a TC antigen was prepared by the same method as in Example 1, 2) from human bladder carcinoma cells. The mice were immunized and also immunized additionally with these antigens in the same way as in Example 1, 3). After the additional immunization, cell fusion was carried out as in Example 1, 4). Antibody-producing cells were selected from the hybridomas by the same method as in Example 1, 5). At this time, the fusion rate was 18.2 %, and the reaction conversion was 14.3 %. The hybridoma which was positive in reactivity was cloned by the same method as in Example 1, 6), and from the resulting hybridoma 6Gl2, a monoclonal antibody was purified by the same method as in Example 1, 7) and 8).The reactivity of the resulting monoclonal antibody was measured in the same way as in Example 1, 9), and the results are shown in Tables 1 to 3.

Table 1 (extracts)

Monoclonal < antibody - 5Fll 6H13 6G12 Type of cells SK-MES-1 0.49 0.01 0.01 Lung carcinoma ME 180 0.43 0.01 0.01 MBI 0.47 0.01 / 0.01 LI-7 0.01 0.46 0.01 Liver carcinoma HuH-7 0.01 0.48 0.01 i BC-4 0.01 0,45 0101 Tumor HC-4 0.01 0.45 0.01 cells HuB-4 0.01 0.01 052 Bladder HUB-15 0.01 0101 0.46 carcinoma Hu-1SN 0.01 0.01 0,48 I HuB-40 ~1 0.01 0.01 0.49 SK-N-F1 0.01 0.01 0.01 Nerve carcinoma SK-N-AS 0.01 0.01 0.01 ( SK-N-DZ 0.01 0101 0.01 Large carcinoma Ovarian carcinolna Table 1 (continued)

Monoclonal ~~~-~~ antibody - 5Fll 6H13 6G12 Type of cells Kidneys ) 0.01 0.01 0.01 Thyroid gland 1 0.01 0.01 0.01 Gallbladder 0.01 0.01 0.01 Leucocyte cells 0.01 0.01 0.01 Placenta 0.02 0.01 0.01 Normal cells Pancreas ~ 0.01 0.01 0.01 Muscle 1 0.01 0.01 0.01 Lungs 1 0.02 0.01 0.01 Liver ; 0.01 0.02 0.01 Colon 0.01 j 0.01 0.01 Intestine 0.01 0.01 0.01 Spleen 0.01 0.01 0.01 Esophagus / 0.01 0.01 0.01 Brain 0.01 0.01 0.01 Bone 0.01 0.01 0.01 HU antigen 0.04 0.05 0.03 (*) The blank value of each cells in EIA was 0.01.

Table 2 (culture supernatant)

Monoclonal - antibody 5Fll 6B13 6612 Type of cells SK-MES-1 0.51 0.01 0.01 1 Lung carcinoma ME 180 0.54 0.01 0.01 MBI 0.51 0.01 0.01 LI-7 0.01 0.49 0.01 Liver carcinoma HuH-7 0.01 0.56 0.01 HC-4 0.01 0.51 0.01 Tumor HC-4 0.01 0.51 0.01 cells BuB-4 Bladder HuB-15 0.01 0.01 0.56 carcinoma Hu-15N 0.01 0.01 0.51 HuB-40 0.01 0.01 0.49 SK-N-F1 0.01 0.01 0.01 Nerve carcinoma SK-N-AS 0.01 0.01 0.01 SK-N-DZ 0.01 0.01 0.01 Large intestine CO-3 0.01 0.01 0.01 carcinoma Ovarian carcinoma NEC 0.59 0.01 0.01 (*) The blank value of each tumor cells in EIA was 0.01.

Table 3 (cells)

Monoclonal antibody 5Fll 6H13 6Gl2 Type of cells SK-MES-1 ++ - - Lung carcinoma ME 180 ++ - MBI ++ - - LI-7 - ++ - Liver carcinoma HuH-7 - ++ - Tumor cells HuB-4 - - ++ Bladder HuB-15 - - ++ carcinoma Hu-15N - - ++ HuB-40 - - ++ SK-N-F1 - - Nerve carcinoma SK-N-AS - - SK-N-DZ - - - Large intestine CO-3 - - - carcinoma Ovarian ++ - - carcinoma NEC Table 3 (continued)

Monoclonal I \ antibody SF11 6H13 6612 Type of cells 1 Kidneys - - Thyroid gland - - Gallbladder ~ ~ ~ Leucocyte - - cells Placenta Normal cells Pancreas ~ ~ ~ Muscle - Lungs - Liver - Colon - Intestine - - Spleen - Esophagus - - Brain - Bone HU antigen + + + Note: + : very strong staining + : weak staining - : not stained COMPARATIVE EXAMPLE 1 1) Preparation of HU antigen 150 ml of a 0.lM phosphate buffer (pH 7.4) containing 0.1 % NP-40 was added to dead EHu (7-10 weeks old) obtained lawfully in accordance with the rules stipulated by Japan Society of Obstetrics and Gynecology.

The mixture was homogenized. The homogenate was stirred overnight at 4 C, and the insoluble components were removed by centrifugation (10,000 rpm, 1 hour). The centrifugal supernatant was passed through a Millipore filter (0.5 / m) (made by Japan Millipore Co.) and used as HU antigen. The protein concentration of the HU antigen was 15 mg/ml.

2) Immunization of BALB/c mice The HU antigen obtained in 1) was intraperitoneally administered to BALB/c mice three times at an interval of two weeks in an amount of 0.1 ml each time.

As a final administration, 0.1 ml of the antigen obtained in 1) was administered to the mice through the vein.

Three days after the final administration, the whole blood was drawn from the mice.

3) Purification of an antibody The blood obtained in 2) was left to stand at room temperature for 2 hours to coagulate it, and converted to the serum. The serum was salted out with 33 z saturated ammonium sulfate three times, and then dialyzed against 0.l5M phosphate-buffered saline (PBS; pH 7.4) to obtain a purified antibody. Its protein concentration was 1.5 mg/ml. The antibody was used after diluting it to 10-fold with PBS.

4) Examination of the reaction specificity of the antibody The followiing TC antigen were provided.

SK-MES-1, ME 180 and MBI derived from human lung carcinoma; LI-7, BuH-7 and HC-4 derived from human liver carcinoma; HuB-4, HuB-15, Hu-lSN and HuB-40 derived from human bladder carcinoma; 5K-N-Fl, SK-N-AS and SK-N-DZ derived from human nerve carcinoma; CO-3 derived from human large intestine carcinoma; NEC derived from human ovarian carcinoma, and also normal human cells.

SK-MES-1 cells were ruptured in 0.1M PBS containing 0.1 % NP-40, and stirred overnight at 4 0C to solubilize and extract them. The extract was centrifuged to remove insoluble components. The extract was then diluted with 0.1M PBS so that its protein concentration became 0.5 mg/ml. The diluted extract was added to a 96-well microplate for EIA at a rate of 50 l per well.

The plate was then incubated at 30 C for 2 hours, and then washed three times with PBS containing 0.05 % Tween 80 (T-80PBS). 0.1 % Bovine serum albumin (BSA) was added at a rate of 50 > q1 per well, and the plate was stored at 4 C (TC antigen-fixed plate). In the same way, tumor cell-fixed plates were prepared by using various cultured tumor cells. The stored TC antigen-fixed plate was washed three times with T-80PBS, and the purified antibody obtained in 3) was added at a rate of 50 l per well. The plate was incubated at 37 C for 1 hour, and then washed three times with T-80PBS. HRP-labelled antimouse IgG antibody was diluted to 1000-fold, and added at a rate of 50 yl per well.The plate was further incubated at 37 C for 1 hour, and washed with T-80PBS three times. A coloring solution containing 60 ml of 0.02 M phosphate-citrate buffer (pH 8.0), 5 l of hydrogen peroxide and 50 mg of ABTS was added at a rate of 100 ss per well to induce coloration. The reaction was stopped with a 0.25 % HF solution, and the absorbance at 510 nm was measured.

The resulting antibody showed reactivity with all the tumor cells and normal cells tested. It was hardly possible to isolate a useful antibody which specifically reacted more with the TC antigens than with the above antibody.

COMPARATIVE EXAMPLE 2 Additional immunization, cell fusion, selection of antibody-producing celles, cloning of hybridomas, preparation of a monoclonal antibody and purification of the monoclonal antibody were carried out in the same way as in Example 1 except that TC antigen prepared as in Example 1, 2) was used instead of the HU antigen used for immunization in Example 1, 3). The resulting monoclonal antibody had no specificity for ovarian carcinoma, but strongly reacted with all tumor-derived TC antigens and normal cells.

REFERENTIAL EXAMPLE Application of the monoclonal antibody (immuno suppressing solid-phase EIA): NEC derived from human ovarian carcinoma and SK-MES-1, ME 180, and MBI derived from human lung carcinoma were provided.

NEC was ruptured in 0.1M PBS containing 0.1 % NP-40 and stirred overnight at 4 0C to solubilize and extract them. The extract was centrifuged to remove insoluble components. The extract was diluted with 0.1 M PBS so that its protein concentration became 0.5 mg/ml.

The diluted extract was added to a 96-well microplate for EIA at a rate of 50/1 per well. The plate was then incubated at 37 C for 2 hours, and then washed with PBS containing 0.05 % Tween 80 (T-80PBS) three times. 0.1 % Bovine serum albumin (BSA) was added to the plate at a rate of 50 yl per well, and the-plate was stored at 4 0C (TC antigen-fixed plate). In the same way as above, tumor cell-fixed plates were prepared by using various cultured tumor cells. The stored tumor cell-fixed plate was washed with T-80PBS three times, and the monoclonal antibody obtained in Example 1, 4) and the serum of a tumor bearing patient or normal human serum were added to the plate at a rate of 50 . The plate was incubated at 37 OC for 1 hour, and then washed three times with T-80PBS. HRP-labelled antimouse IgM antibody was diluted to 1000-fold, and added at a rate of 50 l per well. The plate was incubated at 37 C for 1 hour, and washed three times with T-80PBS. A coloring solution containing 60 ml of 0.02M phosphate-citrate buffer (pH 8.0), 5/ctl of hydrogen peroxide and 50 mg of ABTS was added to the plate at a rate of 100/1. After the coloration, a 0.25 % HF solution was added to stop the reaction. The absorbance at 510 nm was measured, and the reactivity of the antibody was determined. The results are shown in Table 4.

Table 4

Sera of Sera of Sera of Normal patients patients patients human with with lung with benign sera ovarian carcinoma tumors carcinoma Number of posi- 49 42 2 0 tive test subjects Number of nega- 0 0 38 62 tive test subjects Positive 100 100 5 0 rate (%)

Claims (18)

1. We claim: 1. A monoclonal antibody, which (a) reacts with an antigen composed of a sialic acid-containing glycoprotein having a molecular weight of about 600 KD, (b) does not substantially react with human liver carcinoma cells, human bladder carcinoma cells, human nerve carcinoma cells, human large intestine carcinoma cells, human embryonic carcinoma cells, culture supernatants of these cells, and nonionic surfactantsolubilized components of ruptured products of these cells, (c) does not substantially react with normal human kidney cells, normal human gallbladder cells, normal human leucocyte cells, normal human placenta cells, normal human spleen cells, normal human muscular cells, normal human liver cells, normal human colon cells, normal human intestinal cells, normal human pancreatic cells, normal human esophageal cells, normal human brain cells, normal human bone cells, and nonionic surfactant-solubilized components of ruptured products of these cells, (d) does not substantially react with normal human serum, and (e) has such reactivity with an antigen derived from human undifferentiated cells that the absorbance measured by a solid-phase EIA method is about 2 to about 5 times that measured on only the antigen derived from human undifferentiated cells by the same method.
2. 2. The monoclonal antibody of claim 1 of which reactivity with nonionic surfactant-solubilized components of a ruptured product normal human lung cells, normal human ovarian cells or normal human thyroid gland cells is such that the absorbance measured by solid-phase EIA is about 2 to about 5 times that measured on the solubilized components alone by the same method.
3. 3. The monoclonal antibody of claim 1 which is produced by a hybridoma formed by cell fusion of antibodyproducing cells and mouse myeloma cells, said antibodyproducing cells being obtained from mice immunized with an antigen derived from human undifferentiated cells and further with an antigen derived from human ovarian carcinoma cells.
4. 4. The monoclonal antibody of claim 3 in which the antigen derived from human undifferentiated cells is nonionic surfactant-solubilized components of a ruptured product of 7 to 10 week old embryo human, and the antigen derived from ovarian carcinoma cells is nonionic surfactant-solubilized components of a ruptured product of human ovarian carcinoma cells.
5. 5. The monoclonal antibody of claim 1 in which the antigen composed of sialic acid-containing glycoprotein having a molecular weight of about 600 KD is selected from human epithelial ovarian carcinoma cells, human lung carcinoma cells, culture supernatants of these cells, the serum of a human patient with epithelial ovarian carcinoma and the serum of a human patient with lung carcinoma.
6. 6. The monoclonal antibody of claim 1 which belongs to IgM class.
7. 7. The monoclonal antibody of claim 1 in which the hybridoma has the characteristics of hybridoma (FERM BP-1997).
8. 8. A monoclonal antibody which (a) reacts with an antigen composed of i-feto- protein, (b) does not substantially react with human ovarian carcinoma cells, human lung carcinoma cells, human bladder carcinoma cells, human nerve carcinoma cells, human large intestine carcinoma cells, human embryonic carcinoma cells, culture supernatants of these cells, and nonionic surfactant-solubilized components of ruptured products of these cells, (c) does not substantially react with normal humar kidney cells, normal human thyroid gland cells, normal human gallbladder cells, normal human leucocyte cells, normal human placenta cells, normal human spleen cells, normal human muscular cells, normal human lung cells, normal human liver cells, normal human colon cells, normal human intestinal cells, normal human pancreatic cells, normal human esophageal cells, normal human brain cells, normal human bone cells, and nonionic surfactant-solubilized components of ruptured products of these cells, (d) does not substantially react with normal human serum, and (e) has such reactivity with an antigen derived from human undifferentiated cells that the absorbance measured by a solid-phase EIA method is about 2 to about 5 times that measured on only the antigen derived from human undifferentiated cells by the same method.
9. 9. The monoclonal antibody of claim 8 of which reactivity with nonionic surfactant-solubilized components of a ruptured product of normal human liver carcinoma cells is such that the absorbance measured by solid-phase EIA is about 3 to about 5 times that measured on the solubilized components alone by the same method.
10. 10. The monoclonal antibody of claim 8 which is produced by a hybridoma formed by immunizing a mouse with an antigen derived from human undifferentiated cells and additionally with an antigen derived from human liver carcinoma cells, obtaining antibody-producing cells from the mice, and fusing these antibody-producing cells with mouse myeloma cells.
11. 11. The monoclonal antibody of claim 10 in which the antigen from human undifferentiated cells is nonionic surfactant-solubilized components of a ruptured product of a 7 to 10 week old embryo human, and the antigen derived from human liver carcinoma cells is nonionic surfactant-solubilized components of a ruptured product of human liver carcinoma cells.
12. 12. The monoclonal antibody of claim 8 which belongs to IgM class.
13. 13. The monoclonal antibody of claim 10 which has the characteristics of hybridoma FERM BP-2525.
14. 14. A process for producing a monoclonal antibody, which comprises immunizing an animal with an antigen derived from-human undidfferentiated cells, additionally immunizing it with an antigen derived from tumor cells, separating antibody-producing cells from the animal, fusing the antibody-producing cells with myeloma cells to prepare hybridomas, selecting from the hybridomas that hybridoma which produces the desired monoclonal antibody, cultivating the selected hybridoma, and recovering the monoclonal antibody secreted by the hybridoma.
15. 15. The process of claim 14 in which the animal to be immunized is a mouse or rat.
16. 16. The process of claim 14 in which the myeloma cells are myeloma celles derived from a mouse or myeloma cells derived from a human.
17. 17. The prdcess of claim 14 in which the immunization of the animal with the antigen derived from human undifferentiated cells is carried out 3 to 6 times at intervals of 2 to 6 weeks.
18. 18. The process of claim 14 in which the additional immunization with the antigen derived from tumor cells is carried out 1 to 6 weeks after the final immunization of the animal with the antigen derived from human undifferentiated cells.