BR102015032238A2 - HYBRIDOMAS MURINOS PRODUCERS OF HUMAN ANTI-IGM ANTIBODIES - Google Patents

Abstract

The present invention deals with the production of hybridomas for the purpose of applying its product, monoclonal antibodies, in in vitro assays for the diagnosis of infectious diseases, serum protein dosages and other techniques with application in immunohematology. This invention belongs to the modern biotechnology sector. The invention is directed to the use of said hybridomas for the production of human anti-immunoglobulin m (igm) antibodies, which may be used as in vitro assay inputs, as components in the production of diagnostic kits, or used alone as conjugated antibodies ( antibody + enzymes or antibody + fluorochromes) or immobilized on the solid phase of immunoassays (eg polystyrene microplates, nitrocellulose membranes).

Description

Murine hybridomas producing anti-human IgM antibodies FIELD OF THE INVENTION

The invention relates to the production of hybridomas for the purpose of applying its product, monoclonal antibodies, in in vitro assays aimed at the diagnosis of infectious diseases, serum protein dosages and other techniques with application in immunohematology. This invention belongs to the modern biotechnology sector.

The present invention is for the use of said hybridomas for the production of human anti-Immunoglobulin M (IgM) antibodies, which may be used as inputs for in vitro assays, as components in the production of diagnostic kits, or used alone. as conjugated antibodies (antibody + enzymes or antibody + fluorochromes) or further immobilized on the immunoassay solid phase (eg polystyrene microplates, nitrocellulose membranes).

TECHNICAL STATE

Antibodies are molecules composed of four peptide chains, two of which have a molecular weight of approximately 25,000 Daltons (light chains) and the other two have a molecular weight of 50,000 Daltons (heavy chains). (Cellular and Molecular Immunology, 4. Edm Philadelphia: W.B. Saunders, 2000). The finding in 1969 that individuals with B-lymphoid cancers had large amounts of immunoglobulin secreted from a single B-lymphocyte clone in the blood and urine enabled the purification of these myeloma proteins and allowed the individual study of the antibody molecule, determining by first time the complete amino acid sequence of an immunoglobulin (Cellular and Molecular Immunology, 4. Edm Philadelphia: WB Saunders, 2000). These studies demonstrated that an antibody has the ability to bind to a given epitope and discriminate similar epitopes, having two important properties, specificity and diversity, both decisive for the exercise of its defense function and immune surveillance in the body against pathogens. .

All evidence accumulated over the past 30 years makes the antibody a promising agent in the use of human diagnosis and therapy. This and numerous other applications of antibodies run into a major difficulty: the immune system's response to a given antigen is polyclonal. The prospect of isolating a single antibody-producing cell to maintain its in vitro culture is not a viable alternative as they have a limited growth potential, typical of normal somatic cells.

This problem was solved in 1975 by Köhler and Milstein with the discovery of monoclonal antibodies produced by hybridomas, which are cells originated from the in vitro hybridization of antibody-forming somatic cells (B lymphocytes) with continuously replicating cell lines ( myeloma), thus preserving the ability to propagate in culture and to secrete antibodies. The myelomas employed are generally cell lines adapted for in vitro culture from murine developed B-lymphocyte tumors (Eur. J. Immunol. 1975 6: 511-519). Normal antibody-secreting normal B lymphocytes are obtained from mice or rats previously immunized with the antigen of interest. These antibodies have the advantage of being specific to a single antigenic epitope as they are made from a single antibody-producing cell, which makes these molecules free from the problem of cross-reactivity and lack of specificity. Another advantage is the fact that these antibodies can be produced in large quantities, since the producing hybrid cell has the property of immortality (Nature. 1975, 256: 495-497). The process of producing monoclonal antibodies, unlike those for polyclonal antibodies, has the main advantage of not requiring the use of purified antigens. In addition, hybridomas can be stored in liquid nitrogen for later use as needed.

According to Köhler (1981), two advantages of hybridomas are: In the hybrid population, secreted antibodies of choice have a 10 to 50-fold increase over the frequency of production of splenic cells or lymph nodes; and the dominance of myelomas with parenteral characteristics that secrete high levels of antibodies. Myeloma when fused to non-secreting B-lymphomas results in the secretion of the source lymphoma immunoglobulin, that is, the myeloma itself secretes a complete antibody. (In: Immunological Methods. Manchester: Academic Press, 1981,2: 285-308 ).

[007] The continuous culture system enables verification of the influence of physiological and environmental factors affecting cell growth, metabolism and antibody production by hybridomas. However, changes may occur due to long periods in culture and may be of genetic origin, such as point mutations, chromosome loss caused by gene rearrangements, deletion or insertion mutations (Nature. 1977, 27: 299-304, (European Journal 1978, 8: 194-199) may also come from changes in environmental conditions such as pH, temperature, dissolved oxygen or nutritional deprivation (Hybridoma. 1992, 11 (5): 653-664). result in hybridomas with different phenotypic characteristics, including loss of antibody secretory capacity (Hybridoma. 1990, 9: 167-175).

Normally, cells in culture have a sigmoidal proliferative pattern called the growth curve. It represents cellular adaptation to growing conditions, the environment and the availability of substrates and nutrients essential for cell proliferation. The construction of the growth curve is important to evaluate the characteristics of the cells of interest and to establish the cultivation routine, such as the stage at which the cells will be collected and the cell concentration to be cultivated, among other aspects ( How to Grow Cells 1. Rio de Janeiro: Guanabara Koogan, 2005).

The advent of monoclonal antibodies (mAbs) has revolutionized the study of immunology and cell biology. The first monoclonal antibody that identified a T cell subgroup (OKT4, now called CD4) was described by Patrick Kung in 1979 (Science. 1979, 206: 347-349). Currently, these antibodies are extensively used in the laboratory for detection of soluble and cellular antigens in ELISA, IFA, RIA and flow cytometry as components of immunoassay and affinity chromatography kits in the clinic as a therapeutic agent, the benefits of which are still under evaluation, but with enormous prospects of success in the use of cancer treatment, prevention and treatment of transplants, prevention of allergic diseases, carrying drugs to a certain target, basic research, among other applications.

In general the production of monoclonal antibodies begins with the injection of an antigen, which does not need to be purified, usually in the mouse or rat. Within a few weeks, when B lymphocyte proliferation in response to the antigen begins, the animal's spleen, the first source of these lymphocytes, is removed to select these few cells that produce the desired antibodies.

Hybridomas or hybrids can be formed by fusing a lymphocyte cell suspension with continuously replicating tumor cells, such as myelomas or lymphomas, which are fused by rapid exposure to polyethylene glycol. Myeloma cells do not synthesize Hypoxanthine Phosphoribosyl Transferase (HPRT), an enzyme required for the cell to synthesize purines from an extracellular source containing hypoxanthine. The HAT culture medium of these cells contains hypoxanthine and aminopterin, a folic acid-like substance that blocks the endogenous synthesis of purines and pyrimidines, an alternative survival pathway for non-HPRT producing cells. Thus, the replication cell line is selected because of the lack of immunoglobulin production and HPRT activity. At the end of the process, three cell populations remain in culture: splenocytes, myeloma cells and hybrids. Hybrids are selected pending the natural death of splenocytes and myeloma cell line that die from lack of HPRT. Hybrids begin to duplicate every 24-48 hours, with rapid colony formation, as spleen cells supplemented myeloma cells for the production of the HPRT enzyme, which in turn contributed to their immortality property.

Hybridoma cells are then cloned by limiting dilution method, and supernatants tested for antibody production, generally using ELISA, Immunoblot or RIA techniques. A new cloning is performed to ensure monoclonality, where a large number of cells are grown for antibody production. Extensive immunochemical and serological studies are performed to ensure antibody specificity. Selected, cloned and tested hybridomas can be produced indefinitely in mice (ascites) or in culture medium. These cells can also be stored in liquid nitrogen for long term use.

[013] The method of production of mAbs in mice is called ascites induction, this procedure has been a method of choice because it has advantages such as: easy execution, obtaining high concentrations of mAbs, low production cost of each. mg mAbs produced and high success rate to obtain.

Hybridomas represent an important starting point for obtaining highly specific monoclonal antibodies, and it is possible in the culture supernatants of these cells to detect antibodies in amounts ranging from 20 to 100 pg / ml of this protein. This amount may be increased to 1 to 40 mg / ml with intraperitoneal administration of hybridomas in histocompatible or immunodeficient animals that develop an ascites tumor (Appl. Microbiol. Biotechnol. 2002, 59: 135-142). Larger magnification can also be performed in vitro at levels comparable to those obtained in ascites fluid (Res. Immunol. 1998, 149: 532-542) with the possibility of submerged cultivation in bioreactors of these cells, making the supply of monoclonal antibodies produced by the hybridoma.

Therefore, the relevance of the prior production of a hybridoma in order to obtain monoclonal antibodies is evident. Currently there are few hybridomas capable of producing human anti-immunoglobulin. The American Type Culture Collection (ATCC) is the most comprehensive and diverse cell bank in the world, providing about 1000 hybridomas, of which only 21 are human anti-immunoglobulin producers, class or subclass representing only 2, 1% of your collection.

These data underscore the importance of producing new hybridomas with the specificity proposed in this patent, since the monoclonal antibody produced by these hybridomas is widely used in research, diagnosis, and furthermore we have to consider that each hybridoma has specificity and Its own characteristics are capable of producing a specific monoclonal antibody from a large number of epitopes that make up the antigen, thus justifying the existence of inventive activity.

[017] In Brazil, 99% of the inputs used for disease diagnosis or scientific research are imported. Part of these inputs are the antibodies, whose importation implies delay and consequently the reagent when arriving in Brazil may be out of date or damaged by problems of poor packaging during transportation, which leads to loss of money and time. Also, imported products usually have higher cost. In Brazil, spending on these inputs is estimated at around R $ 1.2 billion.

[018] To solve these problems is that in Brazil research and business creation were promoted by different research and development funding agencies. A successful example is the Porto Alegre-based company FK Biotecnologia, which has marketed more than 150 monoclonal antibodies for academic research and develops therapeutic monoclonal antibodies for cancer treatment. The same company has already developed antibody-producing hybridomas against human chorionic gonadotropin for the development of pregnancy diagnosis kits. Another company, Cell B, from the Federal University of Rio Grande do Sul, whose goal is to produce small-volume polyclonal or monoclonal antibodies to serve researchers from Brazil who need an antibody to characterize a newly discovered protein. In the public sphere, one of the first institutions to develop antibodies was the Oswaldo Cruz Foundation (Fiocruz), with the Bio-Manguinhos Monoclonal Antibodies Laboratory in Rio de Janeiro. Since 1983, monoclonal antibodies have been developed for research and diagnostic purposes against specific antigens of public health interest such as hepatitis A, B and C, leptospirosis and yellow fever.

Processes related to hybridoma production are deposited (PI0614475-6; PI0613387-8; PI0212136-0; PI01118234; PI9908466-0; PI9709524-9 and PI9507100-8), none of which produce the monoclonal antibody with the specificity claimed by us.

DESCRIPTION OF THE INVENTION

[020] The present invention relates to a novel hybridoma capable of producing a monoclonal antibody with specificity directed against the human immunoglogulin molecule of the IgM isotype.

[021] The hybridoma of the present invention has enormous application potential because of its ability to expand evenly, massively and permanently. It is capable of producing anti-human IgM monoclonal antibodies without incurring the possible effects of individual response when they are produced in animals, rabbits and goats - polyclonal antibodies - as is commonly done with commercial perspectives. The production process of the present invention allows this to be done uniformly and on a large scale with utility in different in vitro assays with different applications, such as in the dosage of immunoglobulins in human serum, as a reagent in the diagnosis of infectious diseases. In addition, as these are domestic products, they are great options to meet national demand, with lower costs to be purchased, avoiding the need for imports.

Based on the need to produce inputs to supply the national market, we are motivated to make a new hybridoma capable of producing a monoclonal antibody with specificity directed against the human IgM molecule. Our hybridomas enhance the modern national biotechnology with the "in vitro" production of monoclonal antibodies, very specific immune molecules that are of importance in the production of diagnostic kits of different methodologies, such as immunoenzymatic assays, immunofluorescence, immunohistochemistry, radioimmunoassays, assays. precipitation, agglutination, among many others. Anti-IgM monoclonal antibodies have been used extensively in research and clinical analysis laboratories as a very powerful tool for the diagnosis of numerous human diseases, especially infectious ones, being an important tool in determining the phase of a given disease (acute or chronic, primary infection). or secondary), since IgM antibodies are present in the serum in larger quantities in primary or acute infections. For some diseases, it is very important to determine the phase in which it is in order to direct treatment or as prognostic criteria.

[023] The production of "HUMAN IB-IgM HYBRIDOMES" consists of the following steps described in Example I.

Example 1: IMMUNIZATION

[024] It comprises the stage in which mice are immunized with the antigen of interest, so that there is the clonal expansion of the specific B lymphocytes producing the antibodies of interest, which will be used as somatic cells in the production of hybridomas. For this purpose, purified commercial human isotype: IgM (Sigma) immunoglobulins were used as antigen. As an adjuvant we use aluminum hydroxide (Peptgel -Laboratory Teuto Brasileiro S / A). The animals used for immunization were female Balb / c mice (Biotério of the Institute of Tropical Pathology and Public Health - IPTSP of the Federal University of Goiás).

Mice were immunized intraperitoneally with 10 pg IgM and 180 pg Aluminum Hydroxide in a final volume of 500 µl per mouse. The immunization schedule comprised 4 doses at 14-day intervals, and 3 days before sacrificing them to obtain splenic cells, they were challenged following the same conditions as the adopted immunization schedule.

Example 2: PRODUCTION OF HYBRIDOMES

It comprises the step of fusion of myeloma cells SP2 Ag 14/10 with splenic cells of previously immunized mice, resulting in murine anti-human Ig-producing hybridomas, according to the technique of Fasekas et al (1980) (Journal of Immunological Methods 1980, 35: 1-21). Example 3: SELECTION OF HYBRIDOMES

[027] Includes selection of antibody-producing hybridomas of interest from a pool of hybridomas that produce antibodies of different specificities. For this, supernatants from newly produced hybridomas were tested by the in-house ELISA (Enzyme-linked immunosorbet assay) reaction, in which microplates (Corning-Costar Hb) were sensitized with human IgM (the same used in immunizations). To determine the positive samples, the cut-off or reactivity threshold was calculated, calculated as the mean absorbance of the negative controls plus 2 standard deviations. Those non-antibody producing hybridomas that recognize human IgM were then discarded.

Example 4: CLONING

[028] It comprises the step of obtaining and separating different populations by obtaining homogeneous hybrids, that is, originated and replicated from a single mother cell and therefore producing also homogeneous antibodies directed against the same antigenic determinants. Previously selected hybridomas consist of a pool, with antibody-producing cells directed to diverse epitopes, both present in the antigen used in immunization and directed to other distinct epitopes. Thus, this hybridoma pool is polyclonal. For cloning, the limit dilution technique was used. This consisted of obtaining a diluted cell suspension, which was distributed in 96-well culture plates (brand name: Corning Costar) so that we obtained about 1 cell per well. From 5 to 7 days after the start of cloning, the cells were observed under an inverted microscope. The presence of a single cell group characterizes the proliferation of a single clone. Two or more cell groups indicate the presence of multiple clones. About 12-15 days after cloning began, when cells proliferated and covered the entire surface of the well, supernatants were collected from wells that contained only one group of cells to select antibody producing clones of interest. This selection was also made by the same ELISA technique used for post-fusion hybridoma selection. Positive supernatants indicated the presence of antibody producing hybridomas of interest, which are considered monoclonal, as they originated from a homogeneous cell population.

During and after cloning, hybridomas were maintained in HT medium consisting of DMUL culture medium (Dulbecco's Modified Eagle's Medium) (Sigma) supplemented with 15% SFB, 1.36mg / mL hypoxanthine, 388 pg / mL of thymidine, and then kept in simple medium (DMEN supplemented with 10% FBS).

[030] Selected clones were expanded in culture for characterization and stability evaluation.

Example 5: CHARACTERIZATION

It comprises the determination of the light and heavy chain isotype of the antibodies produced by the selected clones. For this an immunoenzymatic assay was performed using the Monoclonal Antibody Isotyping Kit (AP / PNPP) (P \ erce) according to the manufacturer's instructions. In table 1 this data is shown.

Table 1. Characterization of monoclonal antibodies for heavy and light chain isotypes.

Clones Class / Subclass Light Chain Specificity (Heavy Chain) 4B3F8 lgG1 Kappa Anti-lgM

4B3E7 lgG1 Kappa Anti-lgM

4B3D6 lgG1 Kappa Anti-lgM

2C2E3 lgG1 Kappa Anti-lgM

4B3C7 lgG1 Kappa Anti-lgM

Example 6: ASSESSMENT OF CLONES STABILITY, GROWTH CURVE AND ANTIBODY PRODUCTION KINETICS

Assessing the stability of clones comprises the step in which they are tested for maintenance of their ability to produce antibodies. For this, the selected clones were kept in culture for at least 55 days without interruption, in order to evaluate the stability of antibody production in prolonged culture time. Initially, hybridomas were cultured in HT medium and subsequently maintained in simple medium (DMEN supplemented with 10% SFB). After this period, the cells underwent freezing and thawing and were placed in culture to determine the maintenance of the levels of antibodies produced. Supernatants collected during this period were tested by ELISA to confirm that they would maintain antibody production even after prolonged cultivation time. As shown in Table 2, all hybridomas continued to produce high antibody concentrations over the analyzed period. The average O.D. remained high and ranged from 2.84 to 3.05. Ozturk and Pallson (1990) reported that during the time they remain in culture, cells may change due to various factors and changing conditions, whether they are the cells themselves or from the environment. According to the authors, one of the consequences of these changes is the phenotypic differentiation of hybridoma that can result in the loss of antibody secretory capacity, hence the need for periodic stability assessment (Hybridoma. 1990, 9: 167-175). In addition, these hybridomas, after freezing and thawing, were re-cultured and maintained high antibody production.

Table 2. Stability of hybridomas for antibody production over time in culture.

Time in OD Average culture (days) 4B3F8 61 2 ^ 97 4B3E7 59 2.84 4B3D6 59 3.02 2C2E3 55 3.02 4B3C7 61 3.05 [033] Cells have specific characteristics that influence their growth, which also is determined by factors external to cells, such as cultivation conditions and nutrient availability (How to grow cells. 1. ed. Rio de Janeiro: Guanabara Koogan, 2005). Therefore, growth curves are needed to better understand the proliferative profile of the cells of interest.

The cell growth curve comprises the stage at which cell proliferation and death are periodically evaluated in order to obtain a better understanding of the proliferative profile of the cells of interest. For this, 3 days after thawing of the hybridomas of interest, an aliquot of the cell suspension from the original bottles corresponding to 2x10 5 cells was removed, transferred to another bottle and the medium was completed to 5 mL (10% enriched DMEM medium). fetal bovine serum). The curve cells remained in culture for approximately 8 days, during which time the culture medium was not changed. Cells were counted daily on hemocytometer to assess cell concentration and viability was assessed by Trypan blue exclusion method (How to grow cells. 1. ed. Rio de Janeiro: Guanabara Koogan, 2005).

The kinetics of antibody production are made simultaneously to the cell growth curve and comprise the determination of antibody levels produced by such hybridomas periodically. For this, small aliquots of supernatant were collected daily from the curve cells, which were tested by the in-house ELISA method as previously mentioned.

[036] In Figure 1 we have the hybridoma growth curves. As shown, clones 4B3D6 and 2C2E3 reached the maximum cell concentration of 8.6.105 and 1.16.106 cells / mL respectively, in 96 hours, at which time antibody production began to increase. Both showed viability above 90% when they reached the maximum cell concentration, followed by a drop in viability, which coincided with the phase of decline. However, at this stage antibody levels reached the maximum concentration (Figure 2). Clones 4B3F8, 4B3C7 had maximum concentrations of 1.15. 106 and 1.46. 106 cells / mL, time 120 hours, when the viability was high, around 95%. Already 4B3E7, reached the highest cell density, approximately 1.1. 106 cells / mL in 144 hours.

The viability of the 4B3E7, 4B3F8, 4B3C7 hybridomas was approximately 90% over the time period when they obtained maximum antibody production. Clones 2C2E3 and 4B3D6 showed viability of 69% and 75%, respectively, when they achieved higher antibody production (Figures 1 and 2).

[038] These differences confirm de Peres and Curi's (2005) statement that cells have specific characteristics that influence their growth, which is also determined by factors external to cells, such as culture conditions and nutrient availability ( How to Grow Cells 1. Rio de Janeiro: Guanabara Koogan, 2005). Therefore, growth curves need to be constructed to better understand the proliferative profile of the cells of interest.

Example 7: ELISA FOR DETERMINING CLONITY SPECIFICITY

It comprises evaluating the specificity of monoclonal antibodies, ie, which human immunoglobulin molecules (s) the antibodies produced recognize as antigen and are capable of binding. To do so, each was tested against human IgG and IgM.

Microplates (Corning-Costar Hb) were sensitized with 10 µg / ml of the respective human immunoglobulins and incubated at 4 ° C for 12 hours. The plate was blocked with 5% nonfat molar milk in PBS-T (blocking buffer). Hybridoma supernatants were added, as well as positive and negative controls, (respectively sera from mice immunized with the antigen in question and animals inoculated with more adjuvant saline only, diluted 1: 500). Then the peroxidase labeled mouse anti-IgG conjugate (Rabbit anti-Mouse IgG [H + L] HPR Conjugate, Zymed, Invitrogen) was diluted to 1 / 10,000. The chromogen substrate solution used was OPD (0.38 mg / ml o-Phenylenediamine (OPD) (Sigma), 0.003% H2O2 in 0.05M citrate phosphate buffer pH 5.0). Finally, 2N HCl solution was added to stop the enzyme / substrate reaction and absorbances were detected at 492nm using a microplate reader (Thermo). To determine the positive and negative samples, the cut-off or reactivity threshold was calculated, calculated as the mean absorbance of the negative controls plus 2 standard deviations. Positive samples with absorbances higher than the cut-off value plus 10% were considered positive.

As shown in Table 3, said hybridomas produce antibodies specific for human IgM.

Table 3. Test for specificity of monoclonal antibodies tested against human IgM and IgG.

IgM Clones IgG (Cut-off = 0.37) (Cut-off = 0.17) 4B3F8 2Q59 Õ / \ 2 4B3E7 2 ^ 48 Õ33 4B3D6 2JÕ ÕT2C2E3 2 ^ 86 Õ7TÕ 4B3C7 3Í3Í ÕÕ3 Example 8: PRODUCT APPLICATION

[042] It comprises the stage in which antibodies produced by the hybridomas to be patented are tested in the immunodiagnosis in an applied manner, evaluating their ability to recognize human immunoglobulins present in serum, and not only purified and commercial, as already tested in previous reactions. For this we use a commercial ELISA kit (Enzyme Lynked Immunosorbent Assay) by the immunocapture method for detection of anti-Rubella IgM antibodies in human serum (Biokit). Anti-IgM antibodies are usually employed in immunoassay assays in capture methods, ie adsorbed in the solid phase of the reaction (microplate).

[043] The reaction was processed as per manufacturer's instructions. In our tests, however, we replaced the commercial microplate with microplates adsorbed with each of the purified antibodies. The microplates were sensitized with 10 pg / ml antibody in pH 9.6 carbonate buffer, incubated at 4 ° C for 12 hours. In addition, we simultaneously processed a reaction using all kit components, including the microplate, sensitized with anti-human IgM rabbit polyclonal antibody to compare with the results obtained. The concentration of the antibody employed on the commercial plate was not reported by the manufacturer.

For the tests we used previously tested human serum samples positive for anti-Rubella IgM. Table 4 shows as an example one of the reactions made.

Table 4. Monoclonal antibodies used in ELISA reaction for detection of Anti-Rubella IgM antibodies in human serum using commercial kit (Biokit).

Antibodies Monoclonal Results (DO / cutoff) b purified 4B3F8 13 ^ 6 4B3D6 14 ~ Õ 2C2E3 14J 4B3C7 14.0 4Β3Ε7 Ϊ37 Commercial 18.8 aCut-off = CPB x 0.8 (CPB: Low Positive Control Kit) bKit reference values: - Divide the absorbance by the cut-off value. - Positive: DO / cutoff ratio £ 1.0 - Negative: DO / cutoff ratio <0.9 - Doubtful: DO / cutoff ratio £ 0.9 and <1.0 [045] All criteria Reaction validation procedures according to the manufacturer's instructions were observed and met for all antibodies tested.

[046] The results presented in table 4 were satisfactory, since those obtained with our antibodies were similar to the commercial microplate. Thus, these antibodies can be used in immunoassays with different applications, aiming at the detection of human anti-IgM, such as in the diagnosis of infectious diseases.

Murine hybridomas producing anti-human IgM antibodies

Claims (6)

1. Murine hybridomas producing anti-human IgM antibodies characterized by being capable of producing anti-human IgM monoclonal antibodies. 2. Murine hybridomas producing anti-human IgM antibodies characterized by the monoclonal anti-human IgM antibody secreted by hybridoma item 1. 3. Murine anti-human IgM antibody producing murine hybridomas characterized by the process for producing the anti-human IgM monoclonal antibody comprising culturing the hybridoma claimed in item 1 as well as obtaining the antibody produced in the culture supernatant of such cells. 4. Murine hybridomas producing anti-human IgM antibodies characterized by monoclonal antibody produced in ascites following Balb / c inoculation of the hybridomas. 5. Murine hybridomas producing human anti-IgM antibodies characterized by monoclonal antibody produced by submerged cultivation methods that enable the mass increase of hybridoma secreted in vitro monoclonal antibodies. Murine hybridomas producing anti-human IgM antibodies characterized by the use of the hybridoma according to one of the claims 1 to 5, monoclonal antibodies may be produced for use as inputs in the production of diagnostic kits, in assaying for these proteins or as an input used in any other antigen-antibody reactions.