PL234376B1 - Immunoglobulins for agglutination of human erythrocytes, method for obtaining them and applications - Google Patents

Abstract

Immunoglobulins for agglutinating human erythrocytes, in particular an IgG3 antibody capable of specifically agglutinating erythrocytes, in particular a particular blood group, a method for their preparation and use are disclosed.

Description

Description of the invention

According to the information posted on the website of the International Society of Blood Transfusion, 33 blood group systems have been identified so far. Determination of antigens belonging to some systems - especially AB0, Rh, Kell, Duffy and Kidd - is extremely important in cases of blood transfusion, organ transplantation or prevention of serological conflict [1].

Currently, monoclonal antibodies are used to determine the most important blood groups. These are immunoglobulins with a specific amino acid sequence produced in biotechnology laboratories. Two parts are distinguished in the antibody molecule: variable and constant (Fig. 1). The variable part is responsible for binding to the antigen, while the constant part determines the effects that the immunoglobulin can cause upon antigen binding. Due to the type of solid part, there are different classes (isotypes) of antibodies, including IgM, IgG. IgG antibodies are proteins with a molecular weight of about 150 kDa, and the range of their variable parts is no more than 15 nm [2].

Among IgG antibodies, there are four subclasses called isotypes due to the type of heavy chain. Mouse IgG antibodies are divided into IgG1, IgG2a, IgG2b, and IgG3, and their names reflect their electrophoretic mobility. All these isotypes are very similar to each other in terms of structure.

IgM immunoglobulins are much larger, their molecular weight reaches 1300 kDa, and the range of the variable parts exceeds 30 nm [3].

Fig. 1 shows schematically IgG and IgM antibodies. The variable part of the immunoglobulins is shown in white, and the constant part in black. The size scale is kept in the figure. The most important feature of antibodies used in serology is their ability to bind to specific blood group antigens.

The standard technique used in blood group determination is hemagglutination. It consists in mixing the blood sample with a reagent containing monoclonal antibodies directed against the tested antigen. After a few minutes of incubation, it is assessed whether the erythrocytes stick together (agglutinate) as a result of their cross-linking by the antibody used. Agglutination proves the presence of the tested antigen on the surface of red blood cells (Fig. 2). Fig. 2 shows the red blood cell agglutination induced by IgM antibodies. The figure is not to scale, the antibodies are about 1000 times smaller than the erythrocytes.

Erythrocytes suspended in a solution with parameters similar to physiological fluids (e.g. in PBS buffer) have a strong negative charge, which prevents them from coming closer to each other within the range of the variable parts of IgG. Therefore, the above-described hemagglutination reaction can be easily induced by IgM antibodies [4, 5]. These proteins are large enough to easily interact with antigens on two different erythrocytes. Thus, IgM antibodies are very useful in serology because they easily and quickly agglutinate red blood cells.

Currently, the production of IgM antibodies in biotechnology laboratories uses cell lines that:

1. generated after immortalization of murine or human IgM-producing lymphocytes with defined antigen specificity;

2. obtained by means of genetic engineering - they carry in their genome the sequence encoding the IgM class antibody, which has a variable part derived from IgG.

In the first case, the interaction between the produced IgM antibodies and the antigen is described by a dissociation constant greater than for IgG [6]. This means that the binding strength of the variable part to the antigen in an IgM antibody is weaker than that of IgG. The variable parts of IgM antibodies do not undergo somatic hypermutation in nature, which leads to affinity maturation and an increase in interaction with the antigen. The variable parts of IgG antibodies that have undergone a hypersomatic mutation are more antigen-binding, but, as already mentioned, these immunoglobulins are too small to agglutinate red blood cells efficiently.

Many of the obtained monoclonal antibodies that recognize blood group antigens belong to the IgG class. Although their usefulness in diagnostic tests is limited, they can be used in serology by modifying the basic haemagglutination method. For this purpose, red blood cells are subjected to the action of enzymes (eg papain, neuraminidase), which lowers the negative charge on the surface of erythrocytes and may affect the shape of blood cells [7]. Cells with a reduced negative charge become susceptible to agglutination by IgG antibodies. Unfortunately, enzymatic digestion can

PL 234 376 Β1 destroy some antigens - eg belonging to the Duffy group system [8]. In another known method, an additional cross-linking agent is used, the so-called antiglobulin reagent. These are antibodies that recognize immunoglobulin constant parts. The antiglobulin reagent causes agglutination of erythrocytes coated with IgG antibodies. Such a test may not always be performed, especially if the test blood cells are bound to autoantibodies or alloantibodies (eg in some diseases) which will be bound by the antiglobulin reagent and may lead to false positive results.

Genetic engineering can be used to obtain an agglutinating and highly antigenic antibody. The most common method is to obtain an antibody that has an IgG derived variable portion and an IgM constant portion. This solution has been applied many times [9-11], but the production of recombinant IgM causes many problems. In particular, due to their size, oligomerization and rich glycosylation, they are characterized by a low production efficiency [11, 12]. Moreover, no method of controlling the degree of oligomerization of the produced IgMs has been developed so far [9]. In addition, there are observations in the available literature that it is relatively difficult to obtain efficient production of IgM with the variable part derived from IgG [11],

Moreover, unlike IgG, IgM antibodies pose problems in the processing (purification and formulation) stage of the bioproduct. They are relatively unstable, easily aggregate and lose their activity [13], generally cannot be stored in a frozen state [14]. Moreover, the purification requires selecting conditions separately for each IgM antibody, and the obtained purification efficiencies are relatively low [12],

Table 1 summarizes the comparison of the properties of IgG and IgM antibodies.

Table 1

IgG IgM direct agglutination no Yes affinity for the antigen high low molecular weight [kDa] 150 1300 range of variable parts [nm] 15 thirty efficient expression Yes no storage stability Yes no storage possibility in the form of frozen / lyophilisate Yes not / difficult to implement universal methods of cleansing Yes Thread

In summary, the production of IgM immunoglobulins poses numerous problems.

The object of the invention is to provide agents which are suitable for the specific agglutination of erythrocytes, in particular for the determination of blood groups. In particular, it is desirable to provide IgG antibodies capable of direct agglutination of erythrocytes, which would be suitable for the production of improved serological diagnostic reagents. Direct agglutinating IgG antibodies must have absolute specificity and recognize only one type of antigen on the surface of red blood cells, so that they can distinguish erythrocytes from people with different blood groups. Only such antibodies can be used in serological diagnostics. Surprisingly, the present invention has achieved this goal.

The objects of the invention are defined in the appended claims.

One object of the invention is the use of the murine IgG3 antigen-specific antibody from the human ABO system to identify human erythrocytes, preferably the antibody comprises the hinge sequence shown as Seq. no: 7.

Another object of the invention is the use of the murine IgG3 antigen-specific antibody of the human ABO group system in assays based on direct agglutination of erythrocytes, preferably the antibody comprises a hinge sequence shown as Seq. NO: 7. Preferably, in the uses of the invention, a recombinant antibody obtained by incorporating into the structure of a murine IgG3 CDR antibody derived from an antibody with established antigen specificity from a human ABO system, preferably from another class of antibody, for example IgM or IgG, is used.

PL 234 376 B1

During the work on obtaining antibodies recognizing antigens present on human erythrocytes at the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University, numerous immunoglobulins were obtained that recognize blood group antigens from the ABO system. These antibodies are capable of causing a direct agglutination reaction of erythrocytes. Almost all of the antibodies obtained are of the IgM class, except for one, M18G2, which is surprisingly IgG3. The M18G2 antibody, the primary structure of which is shown in Fig. 3, is the first known murine IgG3 antibody that specifically recognizes only the B antigen from the ABO system. It only causes agglutination of erythrocytes expressing B antigen (i.e. erythrocytes of the following blood groups: B, AiB, A2B) and does not cause agglutination of erythrocytes that do not express antigen B (i.e. erythrocytes of the following blood groups: A1, A2, O).

In further studies on agglutinating IgG3 antibodies with anti-B specificity, two different IgG 1 antibodies with anti-B specificity were also genetically engineered, but neither of them, even at high concentrations (10 μg / ml), was able to cause agglutination erythrocytes. This result surprisingly indicates that of the four B-specific IgG subtypes, only IgG3 is capable of causing red blood cell agglutination.

The performed multisequence alignment (MSA) of the amino acid sequences of the individual mouse IgG isotypes: IgG 1, IgG2a, IgG2b and IgG3 indicates a very high degree of similarity of these subclasses. Clearly the differences between them can be seen only in the coding region of the hinge region - ie the fragment of the heavy chain between the CH1 and CH2 domains (Fig. 4). Fig. 4 shows the MSA of the hinge regions of murine IgG antibodies. The cysteine residue that forms the first disulfide bridge connecting the antibody heavy chains is marked.

Murine IgG3 antibodies have the highest number of amino acid residues in the portion of the hinge region preceding the first cysteine residue involved in the formation of a disulfide bridge between heavy chains. The conducted studies on the properties of murine IgG show that the range of variable parts of IgG1 and IgG2b antibodies is similar [2]. Thus, it can be assumed that the differences in the extent of the antigen binding portions are not correlated with the amount of amino acids in the hinge region. However, since the significant differences between the amino acid sequences of murine IgG antibodies are mainly found in the hinge region, it should be considered in the present invention to be the fragment responsible for the erythrocyte agglutination capacity of IgG antibodies.

Example 1. Comparison of biotechnological preparations containing IgM or IgG3 recognizing the B antigen in the direct agglutination test.

Hybridoma cells producing M18 (IgG3), Q6 (IgM) and O10 (IgM) antibodies were seeded at a density of 50,000. cells / ml and grown for 72 h. After completion of the culture, the medium was separated from the cells by centrifugation (200 g for 5 min). The concentration of antibodies in the medium was checked by ELISA method: in the case of IgM antibodies, a commercial antibody kit for the ELISA test (Mouse IgM B Cell ELISpot Development Module, R&D) and the IgM standard (kappa murine IgM, MM-30 clone, Abcam) were used. Plates coated with goat polyclonal antibodies recognizing the murine kappa chain (AbD Serotec) were used to determine the concentration of IgG3 antibodies. Detection was performed with rabbit monoclonal anti-mouse IgG3 antibodies (clone M111-2, Abcam) and horseradish peroxidase-conjugated goat polyclonal antibodies recognizing rabbit immunoglobulins. As a standard, IgG3 purified by protein G affinity chromatography (clone M18) was used, the concentration of which was determined by the bicinchoninic acid method against a bovine serum γ-globulin fraction standard.

The titer of antibodies in biotechnological preparations was assessed in the direct haemagglutination test. Hybridoma cell culture medium was diluted in a two-fold dilution series made in phosphate buffered saline (PBS). On a polypropylene plate intended for slide haemagglutination tests, 100 μl of the antibody containing solutions were mixed with 100 μl of a 5% (hematocrit) suspension of standardized red blood cells, previously washed twice with PBS. By titer was understood the reciprocal of the highest dilution of the preparation that was able to cause agglutination of red blood cells after 5 minutes of incubation at room temperature. The result was assessed macroscopically without the use of optical instruments. Two representative results out of ten experiments are shown.

PL 234 376 Β1

Antibody Titer in the agglutination test Antibody concentration [pg / ml] M18 (IgG3) 64 64 29.4 29.3 Q6 (IgM) 16 16 5.7 5.9 010 (IgM) 32 32 15.7 16.7

Comparison of the agglutination potential of IgM and IgG3 antibodies. The IgG3 antibodies produced by the M18 clone were compared with the IgM antibodies produced by the ΟΊΟ and Q6 clones.

All analyzed antibodies are directed against the B antigen from the ABO group system and differ from one another in variable parts. The O10 and Q6 antibodies have been accepted for commercial use in diagnostics.

Antibody concentration in the analyzed preparations was assessed by ELISA (as previously described). They were then brought to the same molar concentration (3nM) by dilution in a culture medium (DMEM with 10% bovine serum - Lonza) and a series of two-fold dilutions thereof in PBS was made. The ability to agglutinate unmodified reference cells by the prepared samples was demonstrated by the direct haemagglutination test. 100 µl of the antibody solution was incubated at room temperature for 20 min with 0.45% (hematocrit) suspension of group B standard erythrocytes on a 96-well flat-bottomed assay plate. The result of the experiment was assessed using a phase contrast microscope using a five-point scale:

+++, ++, +, +/-, -, ranging from strong to no agglutination reaction.

010 (IgM) Q6 (IgM) M18 (IgG3) Concentration [nM] AND II III AND II III AND II III 3.00 ++ ++ ++ +++ +++ +++ + ++ ++ 1.50 + 4- + 4-h ++ -1-4-4- +/- + + 0.75 + + +/- + + + 0.38 + +/- +/- + + + 0.19 +/- +/- +/- + + + 0.09 - - - +/- +/- +/- 0.05 - - - +/- - - 0.00 - - - - - -

Table - Comparison of the agglutination potential of IgM and IgG3 antibodies. The media from the three cultures of clones 010, Q6 and M18 were diluted so that the antibody concentration was 3 nM and their ability to hemagglutinate human group B erythrocytes was checked.

Example 2. Use of (Fab ') 2 fragments of M18 (IgG3) antibodies for red blood cell agglutination.

mg of M18 antibodies were dialyzed overnight at 4 ° C into pH 4.0 acetate buffer and the sample was concentrated by ultrafiltration (Amicon 100 kDa, Millipore) to a concentration of 0.65 mg / ml. The antibodies were mixed with a concentrated pepsin solution (Sigma) such that the weight ratio of antibody to enzyme was 20: 1. Digestion was carried out for 30 min at 37 ° C, and then the reaction was stopped by increasing the pH to 8.0 by adding 2 M TRIS solution. Antibody fragments were dialyzed into PBS overnight at 4 ° C. The (Fab ') ₂ fragments were purified by negative absorption on a protein A matrix (Pierce). The purity of the prepared preparations was assessed with the use of polyacrylamide gel electrophoresis in the Leammli system. After separation, gels were stained with silver. The complete antibody under reducing conditions travels in the form of two bands corresponding to the heavy chain (50 kDa) and the light chain (25 kDa), and under non-reducing conditions in the form of several bands above 150 kDa. Fragments of (Fab ') ₂ under reducing conditions migrate in the gel in the form of two bands of mass close to 25 kDa, while under non-reducing conditions in the form of a single band corresponding to the 130 kDa protein. Bands corresponding to proteins less than 130 kDa are other products of proteolytic fragmentation, e.g. Fab fragments of about 50 kDa.

PL 234 376 B1

The agglutinating ability of the prepared antibody fragments was assessed in a direct agglutination test. On a polypropylene plate intended for haemagglutination slide tests, 100 μl of solutions containing antibodies or antibody fragments were mixed with 100 μΙ 5% (hematocrit) suspension of standardized red blood cells, previously washed twice with PBS. The result was read macroscopically after 5 min. incubation at room temperature.

The results are shown in Fig. 5, which illustrates the ability of M18 (Fab ') 2 fragments to cause agglutination of group B red blood cells. A - Evaluation of the purity of the prepared antibody fragment preparation. 1 - M18 antibody before proteolytic fragmentation. 2 - (Fab ') 2 fragments obtained after pepsin digestion of M18 antibodies. B - Hemagglutination of group B erythrocytes by M18 antibodies (well 1), fragments (Fab ') 2 of M18 antibodies (wells marked with number 2), negative control (well-).

Example 3. Preparation of recombinant murine IgG3 antibodies capable of a red blood cell glutination.

Changing the class of an antibody to IgG3 is helpful especially in two cases: (i) after obtaining IgG antibodies that specifically bind a specific antigen on the surface of red blood cells, but are not able to agglutinate them; (ii) after obtaining IgM antibodies that are poorly stable and / or difficult to purify. Changing the class of antibodies is done with the use of genetic engineering. The sequences encoding the variable parts of the starting antibody were moved to the "context" of the murine IgG3 and IgG 1 antibodies, which was found on the respective plasmid vectors. This process took place according to the diagram shown in Fig. 6.

RNA was isolated from the hybridoma cells of clones O10 and M18 producing antibodies with anti-B specificity using the Chomczyński method [15] (the O10 antibody is IgM, while the M18 antibody belongs to the IgG3 class). Then, 2 µg of total RNA was transcribed into cDNA using Promega's M-MLV reverse transcriptase according to the manufacturer's instructions. The transcripts encoding the light and heavy chains of the antibodies were amplified using degenerate pairs of primers allowing the amplification of any parts of the variable light and heavy chains [16]. The obtained PCR reaction products were sequenced using the Sanger method (Genomed, Poland). After the light and heavy chain coding sequences were known, primers were designed to allow their cloning into vectors encoding the constant parts of the heavy (pFUSEs-CHIg-mG1, pFUSEs-CHIg-mG3) and light (pFUSE2s-CLIg-mk) (Invivogen) chains.

Primers for the cloning of sequences encoding variable parts:

i. heavy chain of the M18 antibody:

For_M18_HCVF_EcoRI AAGAATTCGCAGGTTCAGCTGCAGCAGTC Rev_Mir_HCVF_AfeI TCCAGCGCTTGAGGAGACTGTGAGAGTG ii. the light chain of the M18 antibody:

For_M18_LCVF_EcoRI CACGAATTCAGATGTTGTTCTGACCCAAACTC Rev_M18_LCVF_BstAPI ATTTAATGCAGCATCTGCCCGTTTTATTTCCAACTTTG iii. the heavy chain of the O10 antibody

For_O10_HCVF_EcoRI AAGAATTCGGAGGAGAAGCTGGATGAGTC Rev_O10_HCVF_Afel TTTAGCGCTTGAGGAGACGGTGACTGAG iv. the light chain of the O10 antibody

For_O10_CLVF_EcoRI CACGAATTCAGATGTTGTGATGACCCAAACTCCAC Rev_O10_CLVF_BstAPI AATTAATGCAGCATCTGCCCGTTTGATTTCCAGCTTG

Cloning into appropriate vectors was performed using restriction endonuclease and ligase according to the enzyme manufacturer's (NEB) instructions. The PCR reaction products containing the heavy chain variable coding sequences (SEQ ID NO. 10) and the pFUSEs-CHIg-mG3 and pFUSEs-CHIg-mg1 vectors were digested with the EcoRl and AfeI enzyme pair. The PCR reaction products containing the variable light chain coding sequences (SEQ ID NO. 11) and the vector pFUSE2s-CLIg-mk were digested with an EcoRI and Bs / API pair. Nucleic acids, after digestion and purification using commercially available kits based on silica columns (Syngen, Poland), were ligated and then transformed into E. coli cells strain Top 10 (Invitrogen) according to the manufacturer's instructions. The correctness of the obtained genetic constructs was verified by Sanger sequencing (Genomed, Poland).

PL 234 376 B1

The plasmid M18_IgG3-pFUSEs-CHIg-mG3 (SEQ. No. 12) encodes the heavy chain of the M18 antibody, the plasmid M18_IgG1-pFUSEs-CHIg-mG1 (SEQ. No. 13) contains the sequence encoding the variable part of the heavy chain of the M18 antibody in the context of IgG1_ and the plasmid M18. -CLIg-mk (SEQ ID NO: 14) encodes the light chain of the M18 antibody.

Plasmids containing the sequences encoding the variable part of the heavy chain of the antibody O10 in the context of IgG3 (O10_IgG3-pFUSEs-CHIg-mG3), the variable part of the heavy chain of the antibody O10 in the context of IgG1 (O10_IgG1-pFUSEs-CHIg-mG1), and the light chain of the antibody O10_pFUSEs CLIg-mk) differed from the plasmids M18_IgG3-pFUSEs-CHIg-mG3, M18_IgG1-pFUSEs-CHIg-mG1 and M18_kappa_pFUSE2s-CLIg-mk only in the fragments encoding the variable part of the light and heavy chains.

Recombinant antibodies were produced in either the mouse myeloma line (NSO, Sp2 / 0) or the human HEK273 line. Cells were transfected using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions to insert the antibody heavy and light chain coding sequences into the genomes. Cells were transfected using 500 ng of DNA and using the following pairs of vectors:

i. to obtain IgG1 antibodies having a variable portion from the M18 antibodies, M18_kappa_pFUSE2s-CLIg-mk and M18_IgG1-pFUSEs-CHIg-mG1 were used, ii. to obtain IgG3 antibodies having a variable portion from M18 antibodies the following were used: M18_kappa_pFUSE2s-CLIg-mk and M18_IgG3-pFUSEs-CHIg-mG3, iii. to obtain IgG1 antibodies having a variable portion from O10 antibodies the following were used: O10_kappa_pFUSE2s-CLIg-mk and O10_IgG1-pFUSEs-CHIg-mG1, iv. to obtain IgG3 antibodies having a variable portion from the O10 antibodies, the following were used: O10_kappa_pFUSE2s-CLIg-mk and O10_IgG3-pFUSEs-CHIg-mG3.

In order to establish stable antibody producing cell lines, cells 72 h after transfection were subjected to selection pressure with two antibiotics: zeocin (400 µg / ml) and blasticidin (8 µg / ml) (Invivogen). The genes for resistance to these antibiotics are encoded on pFUSE plasmids. Cells producing recombinant antibodies were grown in DMEM medium with 10% bovine serum (Lonza).

The transfected cells produced recombinant IgG3 and IgG1 antibodies, the variable parts of which were derived from the O10 and M18 antibodies with specificity for the B antigen from the AB0 blood group system. Antibody concentrations in the culture media were determined by ELISA using murine monoclonal antibody standards (as described in Example 1). For IgG 1 antibodies, purified IgG 1 kappa antibodies purified from the media of MCP21 cell culture (Sigma 96030418) were used as standard. M111-2 antibodies (Abcam) recognize both murine IgG3 and IgG1 antibodies, therefore the concentration of IgG1 antibodies was measured using the same reagents as for IgG3.

The ability of the obtained IgG3 antibodies to agglutinate unmodified blood cells was demonstrated by the direct haemagglutination test. The culture media with known antibody concentration was incubated at room temperature for 20 min with 0.45% suspension of group B erythrocytes in a 96 well assay plate. The test result was assessed using a phase contrast microscope. The agglutination reaction was only observed in wells with IgG3 antibodies present. The use of a similar amount of IgG1 antibodies with the same variable part as the IgG3 antibodies did not lead to agglutination of erythrocytes. The results are shown in Fig. 7. The class change of the starting antibodies was performed according to the presented scheme. The numbers at the bottom of the pictures refer to the antibody concentrations used in the hemagglutination test. Representative result for three independent replicates.

Literature:

1. Dean, L. Blood Groups and Red Cell Antigens 2005.

2. Sosnick, T. R .; Benjamin, D. C .; Novotny, J .; Seeger, P. A .; Trewhella, J. Distances between the antigen-binding sites of three murine antibody subclasses measured using neutron and X-ray scattering. Biochemistry 1992, 31, 1779-86.

3. Czajkowsky, D. M .; Shao, Z. The human IgM pentamer is a mushroom-shaped molecule with a flexural bias. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 14960-5.

4. Bil, J .; Gaciong, Z. Immunohematology. In Immunologia, Gołąb, J .; Jakóbisiak, M .; Lasek, W .; Stokłosa, T., Eds .; 2009; pp. 429-443.

PL 234 376 B1

5. Reid, M. E .; Westhoff, C. M. Membrane Blood Group Antigens and Antibodies. In Blood banking and transfusion medicine Basic principles & Practice, Hillyer, C. D .; Silberstein,

L. E .; Ness, P. M .; Anderson, K. C .; Roback, J. D., Eds .; 2007; pp. 69-79.

6. Malmborg, A. C .; Michaelsson, A .; Ohlin, M .; Jansson, B .; Borrebaeck, C. A. Real time analysis of antibody-antigen reaction kinetics. Scand. J. Immunol. 1992, 35, 643-50.

7. Hyono, A .; Mazda, T .; Okazaki, H .; Tadokoro, K .; Ohshima, H. Analysis of enzyme-treated red blood cell surface and haemagglutination using a theory of soft-particle elecrophoresis. Vox Sang. 2008, 95, 131-6.

8. Westhoff, C. M .; Reid, M. E. Rh, Kell, Duffy, and Kidd Antigens and Antibodies. In Blood banking and transfusion medicine Basic principles & Practice, Hillyer, C. D .; Silberstein, L. E .; Ness, P. M .; Anderson, K. C .; Roback, J. D., Eds .; 2007; pp. 80-95.

9. Gilmour, J. E. M .; Pittman, S .; Nesbitt, R .; Scott, M. L. Effect of the presence or absence of J chain on expression of recombinant anti-Kell immunoglobulin M. Transfus. Med. 2008, 18, 167-74.

10. Huang, T. J .; Reid, M.E .; Halverson, G. R .; Yazdanbakhsh, K. Production of recombinant murine- human chimeric IgM and IgG anti-Js (b) for use in the clinical laboratory. Transfusion 2003, 43, 758-64.

11. Chromikova, V .; Mader, A .; Steinfellner, W .; Kunert, R. Evaluating the bottlenecks of recombinant IgM production in mammalian cells. Cytotechnology 2014.

12. Mader, A .; Chromikova, V .; Kunert, R. Recombinant IgM expression in mammalian cells: A target protein challenging biotechnological production. Adv. Biosci. Biotechnol. 2013, 04, 38-43.

13. Gombotz, W. R .; Pankey, S. C .; Phan, D .; Drager, R .; Donaldson, K .; Antonsen, K. P .; Hoffman, A. S .; Raff, H. V The stabilization of a human IgM monoclonal antibody with poly (vinylpyrrolidone). Pharm. Res. 1994, 11, 624-32.

14. Draber, P .; Draberova, E .; Novakova, M. Stability of monoclonal IgM antibodies freeze-dried in the presence of trehalose. J. Immunol. Methods 1995, 181, 37-43.

15. Chomczynski P .; Sacchi N .; Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr; 162 (1): 156-9.

16. Wang, Z .; Raifu, M .; Howard, M .; Smith, L .; Hansen, D .; Goldsby, R .; Ratner, D. Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3 'to 5' exonuclease activity. J. Immunol. Methods 2000, 233, 167-77.

PL 234 376 Β1

Sequence Listing

Seq. No. 1

M18G2 light chain CDR1

KSLLNSDGFTY

Seq. no: 2

M18G2 light chain CDR2

LVS

Seq. no: 3

M18G2 light chain CDR3

FQSNYLPFT

Seq. no: 4

M18G2 heavy chain CDR1

GYIFSGYW

Seq. no: 5

M18G2 heavy chain CDR2

IFPGSGNT

Seq. no: 6

M18G2 heavy chain CDR3

ARIVPGKYFDC

Seq. no: 7

Mouse IgG3 Hinge Region

EPRIPKPSTPPGSSCPP

Seq. NO: 8 Heavy Chain M18G2

QVQLQQSGAELMKPGASVRISCKATGYIFSGYWIEWTKQRPGHGLEWIGEIFPGSGNTNYKEKFKGKATFT ADTSSNTAYMQLSRLTSEDSAVYFCARIVPGKYFDCWGQGTTLTVSSATTTAPSVYPLGVPGTCVYPLVPVTC

LGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIK RIEPRIFKP3TPPG33CPPGNILGGPSVFIFPPKPKDALMI3LTPKVTCWVDVSEDDPDVHV3WFVDNKE VHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPP REQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPTLDSDGTYFLYSKLTVDTDSWLQGETFTC SVVHEALHNHHTQKNLSRSPGK

Seq. NO: 9 M18G2 light chain

DWLTQTPLSLSVNIGDQASISCKSTKSLLNSDGFTYFDWYLQKPGQSPQLLIYLVSKRFSGVPDRFSGSG SGTDFTLKISRVEAEDLGVYYCFQSNYLPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLN NFYPKDTNVKWKTDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPTVKS FNRNEC

Seq. ID NO: 10 M18G2 heavy chain variable region coding sequence

CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAGGATATCCTGCAAGGC TACTGGCTACATATTCAGTGGCTACTGGATTGAGTGGACAAAGCAGAGGCCTGGACATGGCCTTGAGTGGA GGAGAGAT T T TT T T C CT GGAAGT GG TAATACTAAC TACAAGGAGAAGT CAAGGGCAAGGCCACAT T T T CAC GCAGATACATCATCCAACACAGCCTACATGCAACTCAGCAGACTGACATCTGAGGACTCTGCCGTCTATTT CTGTGCAAGAATCGTACCAGGAAAGTATTTTGACTGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA Seq. ID NO: 11 M18G2 light chain variable region coding sequence

GATGTTGTTCTGACCCAAACTCCACTCTCTCTGTCTGTCAATATTGGAGATCAAGCCTCTATCTCTTGCAA GTCAACTAAGAGTCTTCTGAATAGTGATGGATTCACTTATTTTGACTGGTACCTGCAGAAGCCAGGCCAGT CTCCACAGCTCCTAATATATTTGGTTTCTAAGCGATTTTCTGGAGTTCCAGACAGGTTCAGTGGCAGTGGG CAGGAACAGATT T T T CACAC CAAGATCAGCAGAGT GGAGGC GAGGAT T T T T GGGAGT TGC TAT TAT T CA T C GAGTAACTATCTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGG

PL 234 376 Β1

Seq. no.12

Plasmid M18_IgG3-pFUSEs-CHTg-mG3

GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGG GGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTT TTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGC CGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTG AACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGA GCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCG TTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACCTGAGATCACCGGCGAAGGAGGGC CACCATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGcaggttc agctgcagcagtctggagctgagctgatgaagcctggggcctcagtgaggatatcctgcaaggctactggc tacatattcagtggctactggattgagtggacaaagcagaggcctggacatggccttgagtggattggaga gatttttcctggaagtggtaatactaactacaaggagaagttcaagggcaaggccacattcactgcagata catcatccaacacagcctacatgcaactcagcagactgacatctgaggactctgccgtctatttctgtgca agaatcgtaccaggaaagtattttgactgctggggccaaggcaccactctcacagtctcctcaA GCGCTAC AACAACAGCCCCATCTGTCTATCCCTTGGTCCCTGGCTGCAGTGACACATCTGGATCCTCGGTGACACTGG GATGCCTTGTCAAAGGCTACTTCCCTGAGCCGGTAACTGTAAAATGGAACTATGGAGCCCTGTCCAGCGGT GTGCGCACAGTCTCATCTGTCCTGCAGTCTGGGTTCTATTCCCTCAGCAGCTTGGTGACTGTACCCTCCAG CACCTGGCCCAGCCAGACTGTCATCTGCAACGTAGCCCACCCAGCCAGCAAGACTGAGTTGATCAAGAGAA TCGAGCCTAGAATACCCAAGCCCAGTACCCCCCCAGGTTCTTCATGCCCACCTGGTAACATCTTGGGTGGA CCATCCGTCTTCATCTTCCCCCCAAAGCCCAAGGATGCACTCATGATCTCCCTAACCCCCAAGGTTACGTG TGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCATGTCAGCTGGTTTGTGGACAACAAAGAAGTAC ACACAGCCTGGACACAGCCCCGTGAAGCTCAGTACAACAGTACCTTCCGAGTGGTCAGTGCCCTCCCCATC CAGCACCAGGACTGGATGAGGGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGCCCTCCCAGCCCCCAT CGAGAGAACCATCTCAAAACCCAAAGGAAGAGCCCAGACACCTCAAGTATACACCATACCCCCACCCCGTG AACAAATGTCCAAGAAGAAGGTTAGTCTGACCTGCCTGGTCACCAACTTCTTCTCTGAAGCCATCAGTGTG GAGTGGGAAAGGAACGGAGAACTGGAGCAGGATTACAAGAACACTCCACCCATCCTGGACTCAGATGGGAC CTACTTCCTCTACAGCAAGCTCACTGTGGATACAGACAGTTGGTTGCAAGGAGAAATTTTTACCTGCTCCG TGGTGCATGAGGCTCTCCATAACCACCACACACAGAAGAACCTGTCTCGCTCCCCT GGTAAATGAGCCTAG CTGGCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCT TTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAAC AACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCT CTACAAATGTGGTATGGAATTAATTCTAAAATACAGCATAGCAAAACTTTAACCTCCAAATCAAGCCTCTA CTTGAATCCTTTTCTGAGGGATGAATAAGGCATAGGCATCAGGGGCTGTTGCCAATGTGCATTAGCTGTTT GCAGCCTCACCTTCTTTCATGGAGTTTAAGATATAGTGTATTTTCCCAAGGTTTGAACTAGCTCTTCATTT CTTTATGTTTTAAATGCACTGACCTCCCACATTCCCTTTTTAGTAAAATATTCAGAAATAATTTAAATACA TCATTGCAATGAAAATAAATGTTTTTTATTAGGCAGAATCCAGATGCTCAAGGCCCTTCATAATATCCCCC AGTTTAGTAGTTGGACTTAGGGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCTTCTA GCTTATCCTCAGTCCTGCTCCTCTGCCACAAAGTGCACGCAGTTGCCGGCCGGGTCGCGCAGGGCGAACTC CCGCCCCCACGGCTGCTCGCCGATCTCGGTCATGGCCGGCCCGGAGGCGTCCCGGAAGTTCGTGGACACGA CCTCCGACCACTCGGCGTACAGCTCGTCCAGGCCGCGCACCCACACCCAGGCCAGGGTGTTGTCCGGCACC ACCTGGTCCTGGACCGCGCTGATGAACAGGGTCACGTCGTCCCGGACCACACCGGCGAAGTCGTCCTCCAC GAAGTCCCGGGAGAACCCGAGCCGGTCGGTCCAGAACTCGACCGCTCC GGCGACGTCGCGCGCGGTGAGCA CCGGAACGGCACTGGTCAACTTGGCCATGATGGCTCCTCctgtcaggagaggaaagagaagaaggttagta caattgCTATAGTGAGTTGTATTATACTATGCAGATATACTATGCCAATGATTAATTGTCAAACTAGGGCT GCAgggttcatagtgccacttttcctgcactgccccatctcctgcccaccctttcccaggcatagacagtc agtgacttacCAAACTCACAGGAGGGAGAAGGCAGAAGCTTGAGACAGACCCGCGGGACCGCCGAACTGCG AGGGGACGTGGCTAGGGCGGCTTCTTTTATGGTGCGCCGGCCCTCGGAGGCAGGGCGCTCGGGGAGGCCTA GCGGCCAATCTGCGGTGGCAGGAGGCGGGGCCGAAGGCCGTGCCTGACCAATCCGGAGCACATAGGAGTCT CAGCCCCCCGCCCCAAAGCAAGGGGAAGTCACGCGCCTGTAGCGCCAGCGTGTTGTGAAATGGGGGCTTGG GGGGGTTGGGGCCCTGACTAGTCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCC

PL 234 376 Β1

GTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCATCATGGTAATAGCGATGACT AATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCA TTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGGACTGCCAAGTGGGCAG TTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTC AITATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAAC GCCTGCAGGTTAATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTT GCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACG CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGC CCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG GCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAA AAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTG CAAGCAG CAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTG GAACGAAAACTCACGTTAAGGGATTTTGGTCATGGCTAGTTAATTAACATTTAAATCAGCGGCCGCAATAA AATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGTAACTAACATACGCTCTCCATC AAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTT CTCTATCGAA

Seq. no 13

Plasmid M18_IgGl-pFUSEs-CHIg-mGl

GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGG GGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTT TTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCITCACGCGCCCGC CGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTG AACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGA GCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCG TTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACCTGAGATCACCGGCGAAGGAGGGC

CACCATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCGcaggttc agctgcagcagtctggagctgagctgatgaagcctggggcctcagtgaggatatcctgcaaggctactggc tacatattcagtggctactggattgagtggacaaagcagaggcctggacatggccttgagtggattggaga gatttttcctggaagtggtaatactaactacaaggagaagttcaagggcaaggccacattcactgcagata catcatccaacacagcctacatgcaactcagcagactgacatctgaggactctgccgtctatttctgtgca agaatcgtaccaggaaagtattttgactgctggggccaaggcaccactctcacagtctcctcaAGCGCTAA AACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGG GATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGT

GTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAG CACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAA TTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTC CCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAG CAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAAC CCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTC AATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAA AACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATA AAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGG CAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAA GCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGC ACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGATCCCAGTGTCCCTAGCTGGCCAGA CATGATAAGATACATTGATGAGTTTGGACAAACGACAACTAGAATGCAGTGAAAAAAATGOTTTATTTGTG TLAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACTLAC AACAATTGC ATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATG

PL 234 376 Β1

TGGTATGGAATTAATTCTAAAATACAGCATAGCAAAACTTTAACCTCCAAATCAAGCCTCTACTTGAATCC

TTTTCTGAGGGATGAATAAGGCATAGGCATCAGGGGCTGTTGCCAATGTGCATTAGCTGTTTGCAGCCTCA CCTTCTTTCATGGAGTTTAAGATATAGTGTATITTCCCAAGGTTTGAACTAGCTCTICATTTCTTTATGTT TTAAATGCACTGACCTCCCACATTCCCTTTTTAGTAAAATATTCAGAAATAATTTAAATACATCATTGCAA TGAAAATAAATGTTTTTTATTAGGCAGAATCCAGATGCTCAAGGCCCTTCATAATATCCCCCAGTTTAGTA GTTGGACTTAGGGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCTTCTAGCTTATCCT CAGTCCTGCTCCTCTGCCACAAAGTGCACGCAGTTGCCGGCCGGGTCGCGCAGGGCGAACTCCCGCCCCCA CGGCTGCTCGCCGATCTCGGTCATGGCCGGCCCGGAGGCGTCCCGGAAGTTCGTGGACACGACCTCCGACC ACTCGGCGTACAGCTCGTCCAGGCCGCGCACCCACACCCAGGCCAGGGTGTTGTCCGGCACCACCTGGTCC TGGACCGCGCTGATGAACAGGGTCACGTCGTCCCGGACCACACCGGCGAAGTCGTCCTCCACGAAGTCCCG GGAGAACCCGAGCCGGTCGGTCCAGAACTCGACCGCTCCGGCGACGTCGCGCGCGGTGAGCACCGGAACGG CACTGGTCAACTTGGCCATGATGGCTCCTCctgtcaggagaggaaagagaagaaggttagtacaattgCTA TAGTGAGTTGTATTATACTATGCAGATATACTATGCCAATGATTAATTGTCAAACTAGGGCTGCAgggttc atagtgccacttttcctgcactgccccatctcctgcccaccctttcccaggcatagacagtcagtgactta cCAAACTCACAGGAGGGAGAAGGCAGAAGCTTGAGACAGACCCGCGGGACCGCCGAACTGCGAG GGGACGT GGCTAGGGCGGCTTCTTTTATGGTGCGCCGGCCCTCGGAGGCAGGGCGCTCGGGGAGGCCTAGCGGCCAAT CTGCGGTGGCAGGAGGCGGGGCCGAAGGCCGTGCCTGACCAATCCGGAGCACATAGGAGTCTCAGCCCCCC GCCCCAAAGCAAGGGGAAGTCACGCGCCTGTAGCGCCAGCGTGTTGTGAAATGGGGGCTTGGGGGGGTTGG GGCCCTGACTAGTCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAA ACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCATCATGGTAATAGCGATGACTAATACGTAG ATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTC ATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTA AATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGAC GTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCCTGCAGG TTAATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTA TCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCG TTCAGCCCGACCGCT GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA CGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGGCTAGTTAATTAACATTTAAATCAGCGGCCGCAATAAAATATCTTT ATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGTAACTAACATACGCTCTCCATCAAAACAAAA C GAAACAAAACAAAC TAGCAAAATAGGCT GTCC CCAGTGCAAGT GCAGGT GOCAGAACAT T T C TC C GA TAT A

Seq. no.14

Plasmid M18_kappa_pFUSE2s-CLIg-mk

GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGG GGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTT TTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGC CGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTG AACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGA GCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCG TTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACCTGAGATCAACATGTACAGGATGC AACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACgaattcagatgttgttctgacccaaactcca CTCTCTCTGTCTGTCAATATTGGAGATCAAGCCTCTATCTCTTGCAAGTCAACTAAGAGTCTTCTGAATAG TGATGGATTCACTTATTTTGACTGGTACCTGCAGAAGCCAGGCCAGTCTCCACAGCTCCTAATATATTTGG TTTCTAAGCGATTTTCTGGAGTTCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACAGATTTCACACTCAAG

PL 234 376 Β1

ATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTICCAGAGTAACTATCTTCCATTCACGTT CGGCTCGGGGACAAAGTTGGAAATAAAACGGGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCA GTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAAT GTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAA AGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATA CCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAGAGA CAAAGGTCCTGAGAGCTAGCTGGCCAGACATGATAAGATACATIGATGAGTTTGGACAAACCACAACTAGA ATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTG CAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTT TTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGAATTAATTCTAAAATACAGCATAGCAAAACTTTAA CCTCCAAATCAAGCCTCTACTTGAATCCTTTTCTGAGGGATGAATAAGGCATAGGCATCAGGGGCTGTTGC CAATGTGCATTAGCTGTTTGCAGCCTCACCTTCTTTCATGGAGTTTAAGATATAGTGTATTTTCCCAAGGT TTGAACTAGCTCTTCATTTCTTTATGTTTTAAATGCACTGACCTCCCACATTCCCTTTTTAGTAAAATATT CAGAAATAATT TAAATACATCAT GCAATGAAAATAAAT GT TT T T T TAT TAGGCAG AATCCAGATGCTCAAG GCCCTTCATAATATCCCCCAGTTTAGTAGTTGGACTTAGGGAACAAAGGAACCTTTAATAGAAATTGGACA GCAAGAAAGCGAGCTTCTAGCTTTAGTTCCTGGTGTACTTGAGGGGGATGAGTTCCTCAATGGTGGTTTTG ACCAGCTTGCCATTCATCTCAATGAGCACAAAGCAGTCAGGAGCATAGTCAGAGATGAGCTCTCTGCACAT GCCACAGGGGCTGACCACCCTGATGGATCTGTCCACCTCATCAGAGTAGGGGTGCCTGACAGCCACAATGG TGTCAAAGTCCTTCTGCCCGTTGGTCACAGCAGACCCAATGGCAATGGCTTCAGCACAGACAGTGACCCTG CCAATGTAGGCCTCAATGTGGACAGCAGAGATGATCTCCCCAGTCTTGGTCCTGATGGCCGCCCCGACATG GTGCTTGTTGTCCTCATAGAGCATGGTGATCTTCTCAGTGGCGACCTCCACCAGCTCCAGATCCTGCTGAG AGATGTTGAAGGTCTTCATGATGGCTCCTCctgtcaggagaggaaagagaagaaggttagtacaattgCTA TAGTGAGTTGTATTATACTATGCTTATGATTAATTGTCAAACTAGGGCTGCAgggttcatagtgccacttt tcctgcactgccccatctcctgcccaccctttcccaggcatagacagtcagtgacttacCAAACTCACAGG AGGGAGAAGGCAGAAGCTTGAGACAGACCCGCGGGACCGCCGAACTGCGAGGGGACGTGGCTAGGGCGGCT TCTTTTATGGTGCGCCGGCCCTCGGAGGCAGGGCGCTCGGGGAGGCCTAGCGGCCAATCTGCGGTGGCAGG AGGCGGGGCCGAAGGCCGTGCCTGACCAATCCGGAGCACATAGGAGTCTCAGCCCCCCGCCCCAAAGCAAG GGGAAGTCACGCGCCTGTAGCGCGAGCGTGTTGTGAAATGGGGGCTT GGGGGGGTTGGGGCCCTGACTAGT CAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACG CCCATTGATGTACTGCCAAAACCGCATCATCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAG TAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATA GGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCA TTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGG GGTCGTTGGGC GGTCAGCCAGGC GGGCCATT TACC GTAAGT TAT GTAAC GCCT GCAGGT TAAT TAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTC CGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTG TAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGA TTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGAACAGTATTTGGTATCTGCGCTCTGGT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC C GGCAAACAAACCACCGCT GGTAGC GGTGGT TT T T T T TTGT GCAAGCAGCAGATTACGC GCAGAAAAAAAG GATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG ATTTTGGTCATGGCTAGTTAATTAACATTTAAATCAGCGGCCGCAATAAAATATCTTTATTTTCATTACAT CTGTGTGTTGGTTTTTTGTGTGAATCGTAACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAA ACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCAGGTGCCAGAACATTTCTCTATCGAA

Claims (13)

Patent claims 1. An isolated antibody or antigen-binding fragment thereof, comprising: a. A light chain variable domain (VL) comprising: i. the light chain CDR1 sequence according to SEQ. no: 1, ii. the light chain CDR2 sequence according to SEQ. no: 2, iii. the light chain CDR3 sequence according to SEQ. no: 3 PL 234 376 B1 and b. A heavy chain variable domain (VH) comprising: i. the heavy chain CDR1 sequence according to SEQ. no: 4, ii. the heavy chain CDR2 sequence according to SEQ. no: 5, iii. the heavy chain CDR3 sequence according to SEQ. ID NO: 6, wherein said antibody or antigen binding fragment thereof binds specifically to antigen B from the human ABO system. 2. The antibody or fragment thereof according to claim 1 The method of claim 1, comprising the hinge sequence shown as Seq. no: 7. 3. The antibody or fragment thereof according to claim 1 The method of claim 1, wherein the antibody is a murine IgG3 type antibody. The antibody fragment of claim 1 The method of claim 1, which is the (Fab ') 2 fragment of a murine IgG3 antibody. The antibody or fragment thereof according to claim 1 The antibody of claim 1, wherein the heavy chain of the antibody has the amino acid sequence shown as Seq. ID NO: 8 while the antibody light chain has the amino acid sequence shown in SEQ. no: 9. 6. Nucleic acid sequence encoding the antibody or fragment thereof as defined in claim 1 from 1 to 5. 7. A method for the preparation of an antibody or a fragment thereof capable of agglutinating human erythrocytes, characterized in that a) a nucleic acid molecule encoding the antibody light chain that binds specifically to the human erythrocyte is isolated, b) the fragment encoding the VH variable domain or its fragments encoding CDR regions is isolated from the nucleic acid molecule encoding the heavy chain of the antibody that specifically binds to the human erythrocyte, c) the fragments obtained in step b) are replaced with the corresponding fragments encoding the VH variable domain or its fragments encoding the CDR regions contained in the nucleic acid molecule encoding the heavy chain of the murine IgG3 antibody or its fragment being part of (Fab ') 2, d) culturing the host cell transfected with the coding sequences obtained in steps a) and c), and then isolating from the culture broth an antibody capable of agglutinating human erythrocytes or its fragment (Fab ') 2, e) wherein the antibody or fragment thereof produced is an antibody or fragment thereof as defined in claim 1; from 1 to 5. 8. A method for the preparation of an antibody or a fragment thereof capable of agglutinating human erythrocytes, characterized in that a) a nucleic acid molecule encoding the antibody light chain that binds specifically to the human erythrocyte is isolated, b) a nucleic acid molecule encoding the heavy chain or its fragment being part of the (Fab ') 2 antibody that binds specifically to the human erythrocyte is isolated, c) in the nucleic acid molecule obtained in step b), the hinge coding fragment is replaced with a sequence coding for the hinge region of the murine IgG3 antibody according to Seq. no.7, d) culturing the host cell transfected with the coding sequences obtained in steps a) and c), and then isolating from the culture broth an antibody capable of agglutinating human erythrocytes or a fragment thereof (Fab ') 2, the produced antibody or fragment thereof being an antibody or a fragment thereof as defined in claim from 1 to 5. 9. A composition for identifying human group B erythrocytes comprising the antibody or fragment thereof as defined in claim 1; from 1 to 5. 10. A composition for agglutinating human erythrocytes comprising the antibody or fragment thereof obtained by a method as defined in claim 1, from 7 to 8. 11. The composition according to p. The method of claim 10 for the agglutination of human group B erythrocytes, comprising the antibody or fragment thereof as defined in claim 1, from 1 to 5. PL 234 376 Β1 12. The use of the murine IgG3 antigen-specific antibody of the human ABO system for the identification of human erythrocytes, wherein the antibody used is the antibody of claim 1. from 1 to 5. 13. Use of the murine IgG3 antigen-specific antibody of the human ABO group system in an assay based on direct agglutination of erythrocytes, wherein the antibody comprises the hinge sequence shown in Seq. NO: 7, preferably the antibody used is an antibody according to claim 1. from 1 to 5. Drawings > 30 nm IgM Fig. 1 Fig. 2 PL 234 376 Β1 M18G2 heavy chain amino acid sequence QVQLQQSGAELMKPGASVRISCKAT GYIFSGYW LEWTKQRPGHGLEWIGE IFPGSGNT NYKEKFKGKATFT> FR1> CDR1> FR2> CDR2> FR3 ADTS SNTAYMQLS RLT S EDSAVYF RRIVPGKYFDC WGQGTTLTVSSATTTAPSVYPLVPGCSDTSGSSVT> CDR3> CH1 LGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIK RIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVWDVSEDDPDVHV3WFVDNKE> Hinge> CH2 VHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTTPPP> CH3 REQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTC SVVHEALHNHHTQKNLSRSPGK * M18G2 kappa light chain amino acid sequence DWLT QT PL SLS VbII GDQA SI SC KS T KSLLNSDGFTY FDWY LQ KP GQ S PQLLIY KRFSGVPDRFSGSG> FR1> CDR1> FR2> CDR2 S G T D F T L KIS R V E AE DL G V Y Y C FQSNYLPFT FGSGTKLEIK.RADAAPTVSIFPPSSEQLTSGGASWCFLN> CDR3> CL NFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC * Fig. 3> IgG3 EPRIPKPSTPPGSS§PP --------> IgGl -------- VPR _D CGgXP _C ICTV ---> IgG2a EPRGP - T --- TRP ^ PPCKC ---- PA> IgG2b EPSGPIST --- INPgpPCKECHKCPA Fig. 4 PL 234 376 Β1 Fig. 5 PL 234 376 Β1 3. Sequencing of traascripts encoding the heavy and light chains of the starting antibody 4. Designing the starters _____________ _{_} ________ Λ *. * A *. * A * AM> AAAAAAAAAAAA-3 ' Fig. 6 PL 234 376 Β1 Fig. 7 Antibody Ml8 IgG3 329 ng / ml 378 ng / ml