An erythrocyte-specific antibody screening test kit and preparation method of this test kit

Abstract

The invention relates to an erythrocyte specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen (RhD and RhCE as 2 different antigens derived from RBC and 45 or more antigens in total including some variants of these two antigens) and a preparation method of this test kit. Said antigens are transgenically produced and provide the clearest results compared to traditional methods of antibody screening. Field-targeted mutation process is applied during the preparation of the erythrocyte-specific antibody screening test kit subject to the invention.

Description

[0001] DESCRIPTION

[0002] AN ERYTHROCYTE-SPECIFIC ANTIBODY SCREENING TEST KIT AND PREPARATION METHOD OF THIS TEST KIT

[0003] Technical Field of the Invention

[0004] The invention relates to an erythrocyte-specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen and a preparation method of this test kit. Said antigens are transgenically produced and provide the clearest results compared to traditional methods of antibody screening.

[0005] State of the Art

[0006] Y-shaped proteins that are part of the immune system and fight foreign substances in the body are called "antibodies" or "immunoglobulins". These foreign substances, known as antigens, can be any bacteria, viruses, or chemicals. Each type of antibody produced for foreign substances is unique and is used to recognize the structure on the antigen. Thus, antibodies bind to antigens to remove them from the body and form complexes for the immune system.

[0007] In the state of the art, detection of antibodies to blood group antigens is crucial in pre- and post-transfusion testing to ensure sufficient and reliable blood supply to patients requiring blood transfusions. An antibody test, called a "serology test", is the process of determining the presence or amount of a specific antibody in a blood sample taken from a person. Some antibody tests focus on a specific type of antibody. For example, the most common antibody tests are IgM, IgG, and IgE. The purposes of antibody tests are to evaluate the immune status against a specific microorganism, to monitor the course of a specific infection, to evaluate exposure to an infectious antigen, to investigate the causes of transfusion reaction or rejection of a transplanted organ by the body, and to diagnose problems caused by allergies, hepatitis, HIV, liver diseases, syphilis, lupus, thyroid, or autoimmune disorders[1]. In the present art, the most serious complications of blood transfusion result from interactions between antibodies in the recipient's plasma and surface antigens on donor erythrocytes. Each surface antigen has different levels of immunogenicity. ABO, Rhesus D, RhCE, and Kell antigens have the highest immunogenicity. Patients who require regular blood transfusions may develop antibodies against foreign donor antigens, resulting in hemolytic reactions that can be fatal for the patient. Therefore, it is crucial to screen the patient for antibodies before transfusion.

[0008] Antibody screening tests in the present art are based on the interaction of erythrocyte cell suspensions of known antigen phenotype with recipient serum. Using the antigen panel listing the known antigens of commercial erythrocytes, the erythrocytes to which the patient's serum reacts positively and negatively are identified. The common surface antigens of the erythrocytes that react positively are determined from the antigen panel and then it is determined that the patient has antibodies against said antigen. However, a detection method based on an elimination method using a limited antigen panel does not provide a precise result that can guarantee a patient test result. It is not possible to determine exactly which of the antigens with a common positive reaction interacts with the antibodies. Antibodies developed against erythrocyte antigens may be present in the serum free and / or coated on the surface of erythrocytes. Free circulating antibodies are detected by Indirect Coombs test. Indirect Coombs test (antibody screening test) is performed in recipients and pregnant women before transfusion. In said test, antibodies are identified in all positive cases and identification tests are performed with commercially available human erythrocyte suspensions of known phenotype using the gold standard gel centrifugation method. However, there are difficulties in evaluation due to the presence of multiple antigens on the erythrocyte surface. If autoantibodies or alloantibodies to high-prevalence blood group antigens are present in the patient's blood group sample, clear identification can be problematic due to non-specific positive (false positive) or overlapping reactions on antibody screening. The results of these antibody tests performed before transfusion with 2-, 3- and 4-cell screening panels and 11 -cell panels cannot always be interpreted correctly. Difficulties arise during the evaluation phase due to the presence of multiple antigens on the red blood cell membrane. It is often impossible to distinguish precisely which antigen / antibody is responsible for a positive result. For example, in the study conducted with cell panels containing both Kell blood group system antigens K and k antigens and Duffy blood group antigens Fya and Fyb antigens on the erythrocyte (RBC) surface, it is impossible to distinguish whether the positive reaction is due to the presence of Kell antibodies or Duffy antibodies in the patient serum / plasma. In the patent document numbered EP2298785A1 in the state of the art, there is an analysis method for making antigen / antibody detection safe. In the invention described in said patent document, a multiplex-based test system is created using individual SNPs. Said test system detects antigens or antibodies in patient / donor blood with a fluorescent radiation measuring device. In said method of analysis, the test result can only be evaluated by means of an instrument reading. This leads to an increase in the cost of test kits.

[0009] Reasons such as the limitations and insufficiencies of the antibody screening test kits in the state of the art, the inability of the antibody screening test kits in the state of the art to provide a precise result that can guarantee a patient test result due to the fact that they work based on a detection method based on a elimination method using a limited antigen panel, the inability of said antibody screening test kits to determine exactly which of the antigens for which a common positive reaction is obtained interacts with the antibodies, the difficulties in antibody screening due to the presence of more than one antigen on the erythrocyte surface during the antibody evaluation phase, the difficulties in antibody screening due to the presence of more than one antigen on the red blood cell membrane during the evaluation phase, and the impossibility of distinguishing precisely which present antigen / antibody is responsible for the positive result have made it necessary to introduce an antibody screening test kit that eliminates all of the problems mentioned.

[0010] Summary and Objects of the Invention

[0011] The invention discloses an erythrocyte specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen (RhD and RhCE as 2 different antigens derived from RBC and 45 antigens in total including some variants of these two antigens) and a preparation method of this test kit. Said antigens are transgenically produced and provide the clearest results compared to traditional methods of antibody screening.

[0012] An object of the invention is to provide an erythrocyte-specific antibody screening test kit for antibody screening, which provides the most accurate results. The antibody screening tests performed with erythrocyte cell suspensions in the state of the art without reaching a definitive conclusion based on the antigenic panel, are insufficient for the precise and accurate detection of the patient’s antibodies. While preparing the erythrocyte-specific antibody screening test kit subject to the invention, genetic modifications are made on cell lines in cell culture in vitro and cells expressing a single antigen from target erythrocyte (RBC) antigens are obtained. By way of example, in the state of the art, in the study conducted with cell panels containing both Kell blood group system antigens K and k antigens and Duffy blood group antigens Fya and Fyb antigens on the erythrocyte (RBC) surface, it is impossible to distinguish whether the positive reaction is due to the presence of Kell antibodies or Duffy antibodies in the patient serum / plasma. Since only K or k antigens belonging to the Kell blood group system antigens will be carried at one time while preparing the erythrocyte-specific antibody screening test kit subject to the invention, it will be possible to accurately determine the presence of antibodies in the patient serum. Molecular biology and genome editing processes are applied in the preparation method of the test kit subject to the invention. HEK293 cell line and / or CHO cells are used in the preparation of the test kit subject to the invention and RBC antigens are not included in the cell membranes of CHO cells. While preparing the erythrocyte-specific antibody screening test kit, the targeted antigens will be described using the HEK293 cell line, which can rapidly proliferate in cell culture and can be easily transfected. Said antigen systems are ABO, MNS, P1 PK, Rh (RH), Lutheran (LU), KEII (KEL), Lewis (LE), Duffy (FY), Kidd (JK), Diego (DI), Yt (YT), Xg (XG), Scianna (SC), Dombrock (DO), Colton (CO), Landsteiner-Wiener (LW), Chido / Rodgers (CH / RG), H, Kx (XK), Gerbich (GE), Cromer (CROM), Knops (KN), Indian (IN), Ok (OK), Raph (RAPH), JohnMiltonHagen (JMH), I, Globoside (GLOB), Gill (GIL), Rh-associated glycoprotein (RHAG), FORS, JR, LAN, Vel (VEL), CD59, Augustine (AUG), Kanno (KANNO), SID, CTL2, PEL, MAM, EMM, ABCC1 , Er (ER), and CD36. In the present art, blood transfusions can be performed if the results are suitable between the recipient and the donor after the suitability tests performed before blood transfusion in the Immunohematology laboratories in the Blood Centers. After ABO blood grouping, the most important tests are crossmatch and antibody screening / identification tests. Antibody identification tests contain a certain concentration of human blood of known phenotype. The patient's blood (plasma) is tested with these cells to determine the presence and / or absence of antibodies. In the specific antibody screening test kit subject to the invention, the antibody identification cells will be cells specifically designed with genetic modification; in other words, they will be cells containing only one phenotype. In this way, a cell that is positive in a patient sample will be reported as positive for that phenotype and a cell that is negative will be reported as negative for that phenotype. The ultimate objective of the test kit subject to the invention is that the cells obtained specific to each antigen can be used instead of 2, 3 and 4-cell screening panels and 11 -cell identification panels. The antibody screening tests performed with erythrocyte cell suspensions used in the state of the art without reaching a definitive conclusion based on the antigenic panel are insufficient for the precise and accurate detection of the patient’s antibodies. These tests are performed using commercially available 2-, 3-, 4-screening and 11 - identification erythrocyte cell suspension panels. In the test kit subject to the invention, besides the screening of serum antibodies, the production of antigens targeting the screening of other antibodies (bacterial, viral infections, etc.) in case of need, provides the clearest results in antibody screening.

[0013] In the test kit subject to the invention, results can be obtained with simple test techniques such as gel centrifugation method or slide method without the need for any extra device etc. system. In the test kit subject to the invention, the patient sample is studied with an antibody screening-identification cell to be produced using a coombs card containing a dextran-derived gel. After incubation for 10-15 minutes at 35-37 C, the results are evaluated by their severity from +- to 4 positivity.

[0014] Detailed Description of the Invention

[0015] The invention relates to an erythrocyte specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen (RhD and RhCE as 2 different antigens derived from RBC and 45 or more antigens in total including some variants of these two antigens) and a preparation method of this test kit. Said antigens are transgenically produced and provide the clearest results compared to traditional methods of antibody screening. Field-targeted mutation process is applied during the preparation of the erythrocyte-specific antibody screening test kit subject to the invention. The Hek293 cell line and / or CHO cells to be manipulated within the scope of the invention do not express antigens of the target red blood cells. In the invention, gene sequences related to lentiviral-based plasmids are transferred to cells, enabling the expression of antigens.

[0016] In the test kit subject to the invention, the relevant antigens are cloned into the pLenti- lll-HA (lentiviral) vector to ensure stable and overexpression in HEK293 and / or CHO cell lines. Complementary DNAs (cDNAs) of the target antigen are commercially available. Using commercially available cDNAs as templates, the target gene is amplified with the highly accurate PrimeStar DNA polymerase (Takara, Clontech) according to the manufacturer's instructions. The quality control (yield, specificity) of the obtained PCR products (insert) is performed on agarose gel. After ensuring the quality, PCR products are obtained from the gel according to the manufacturer's protocol (MN Gel Extraction, PCR Clean-up Kit). The resulting PCR products and the pLenti-ll l-HA vector are digested with the relevant restriction enzymes (Thermo / NEB / Takara etc.) as recommended by the manufacturer. The digestion products are extracted from the agarose gel according to the manufacturer's protocol. Ligation of the PCR products obtained as digested to the digested pLenti-l I l-HA vector is carried out with the T4 ligase (Takara) enzyme following the manufacturer's protocol. Transformation of the ligation products into competent E. coli DH5alpha bacteria by heat shock is carried out. Agar medium containing the relevant resistance antibiotic are inoculated, it is incubated at 37°C for a minimum of 16 hours and colonies are observed. The obtained colonies are firstly screened by colony PCR to find the colonies containing the targeted plasmid and the PCR products obtained are checked in agarose gel and positive colonies (at least two colonies for each target clone) are inoculated into liquid medium for plasmid isolation. Inoculated colonies were incubated on a 37°C shaker for a minimum of 16 hours. Plasmid isolation is then performed using a mini isolation kit. The isolated plasmids are treated with the restriction enzymes used for cloning to determine whether the correct clones have been obtained. The digestion products are checked in agarose gel. The same size is obtained as a result of enzyme digestion of the relevant gene transferred into the vector in the same way. Red blood cell surface antigens are highly polymorphic and a single base change can cause antigen alteration. Therefore, plasmids that express other antigens are obtained by site-targeted mutation. For example, among the RHCE group antigens "C" on the erythrocyte surface, changing the thymine base at location 307 to a cytosine base converts the serine at position 103 to a proline amino acid, resulting in the formation of the "c" antigen and a vector expressing "c" antigen can be obtained by performing site- targeted mutation on the vector obtained for C antigen expression. Using the designed primers and QuickChange II Site Targeted Mutation kit (Agilent), plasmids are generated by whole plasmid mutagenic PCR following the manufacturer's protocol to obtain antigens expressed as a result of one or more nucleotide changes. Briefly, the mutant PCR product obtained using the mutation primers is treated with Dpnl restriction enzyme to destroy the parent plasmid (so that only the mutated DNA strand is present) without any further treatment, and the resulting digestion product can be transformed into a suitable competent bacterium. HEK293 and / or CHO cells are rapidly thawed at 37 °C in approximately 2 minutes, it is mixed with 9 ml of medium and centrifuged for 5 minutes at a centrifugation speed of 125x g. The resulting pellets are resuspended in the manufacturer's recommended medium (Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for HEK293, F-12K medium supplemented with 10% FBS for CHO) and seeded into a 75 cm2flask. Cells are incubated under 5% CO2 and collected before reaching 90% density. After the initial culturing, the growing cells need to be sub-cultured for stocktake and preparation for studies. For this, the medium is carefully removed from the environment. Cells are exposed to 0.25% Trypsin-0.5 mM ethylene diamine tetraacetic acid (EDTA) solution and removed from the surface to which they are attached. Trypsin-EDTA activity is stopped by adding medium on the cells. For cells to be stocked in a liquid nitrogen tank, 5% dimethyl sulphoxide (DMSO) is added to the medium. Cell density is determined by cell counting with trypan blue. As many cells as needed are diluted and used or stock is taken. The resulting pLenti-lll-HA vectors are lentivirally packaged to be transduced for antigen expression in HEK293 and / or CHO cells. The preferred cell line for lentiviral packaging is HEK293 cells. The relevant vectors are added together with the packaging vectors psPAX2 (Addgene) and pCMV- VSV-G (Addgene) to the medium of the HEK293 cell line at 90% density in a 10 cm petri dish and the medium is replaced with fresh medium after 16 hours. The first virus collection (medium of the cell) is performed in the first 24 hours following the medium change, and the second virus collection is performed in the 48th hour. The virus suspension obtained is transferred into 2 mL tubes and stored at -80°C. Lentiviral packages are introduced into HEK293 cells and / or CHO cells and the desired erythrocyte surface antigen expression is performed. For this purpose, lentiviral particles are introduced to HEK293 and / or CHO cells in separate cultures. In a 6-well petri dish, transduction is performed with polybrene (hexadimethrin bromide) into cells at 80% density. The lentiviral particles stored at -80°C are brought to room temperature and homogenous dispersion of the particles is ensured using a pipette by adding polybrene. The medium is removed from the cell and lentiviral particle suspension is introduced. The medium is refreshed 16 hours after transduction. In order for the transduced plasmids to be permanently expressed in the cell, cells that have not received the virus particle must be eliminated. Therefore, antibiotic selection is initiated in the cell following the medium change. Puromycin is used because the IpLenti-l ll-HA vector backbone contains a puromycin resistance gene. The puromycin concentration to be used is used as the value obtained from the "killing curve". At the end of approximately 1 week of selection, a polyclonal cell population stably expressing the desired plasmid is obtained. Since the obtained cells will express the gene at different levels, in order to counter the possibility of an increase over time in cells with low gene expression, the cells in the resulting polyclonal cell line is diluted according to the ‘limiting dilution’ Addgene protocol, calculated so that 0.5 cells fall per well, and seeded into a 96-well petri plate. Thus, a monoclonal cell line is obtained by dropping a single cell into each well. These cells are grown and their expression levels are analyzed, and cells with no expression or expression levels below 10% are selected. Whether the obtained monoclonal cell lines express the desired protein is confirmed by protein immunoblot (Western Blot) method. It is important that adherent cells retain this property for easy culturing and easy genetic manipulations. However, it is thought that suspending these cells at the end of the targeted genetic changes will facilitate their growth and adaptation to the gel centrifugation method. The possibility of aggregate formation, which can become one of the biggest problems in gel centrifugation adaptation, can be minimized by using cells in suspension. Suspension of HEK293 and / or CHO cells is performed by serum dilution method. Reducing the amount of FBS over time from medium containing 10% FBS, which is the normal growth medium of these cells, causes the cells to lose their adherent properties. Cells removed by trypsin- EDTA from the culture medium they are grown in are collected by centrifugation and resuspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% FBS. Cells are grown until they reach maximum cell density. The viability of cells reaching maximum density is examined by staining with Trypan blue. If the viability rate is above 85?%, the cells are considered to be acclimatized to this medium. The same procedures are repeated, each time using a lower concentration of FBS. For HEK293 cells, 2.5%, 1%, 0.5% and finally 0.1% FBS was added to acclimatize the cells to this low serum medium. For CHO cells, medium with 2.5%, 1%, 0.6% and finally 0% FBS (without serum) is used respectively. When switched to serum-free medium for CHO cells, the medium is supplemented with pluronic additive. If cell viability falls below 80% at these stages, it is continued after repeating the adaptation process by returning to the upper serum level. In this process, the ratio of the amount of suspended cells to the amount of adherent cells increases and as a result of the process, suspended cells are obtained. After obtaining the suspended cells, the cells are grown by incubating 5% CO2in a shaking incubator (50-80 rpm). The modified cells used in the test kit subject to the invention need to be adapted to the agglutination method in order to be used in antibody screening and identification test. Factors such as the gel material used in the gel centrifugation method, the pore size formed, and the solution in which the gel is swollen are important for obtaining the best result. At this stage, experiments were performed with gel matrices containing particles of various sizes and structures (Sephadex (G-25 fine, G-50 superfine special grade, G-50 DNA grade, G50 medium, G-75, G-100), Dextran (G-50), Sephacryl 200, Zetadex 50, Agarose (4% BCL, 6% BCL, 10% BLC)). HEK293 and / or CHO cells stained with each gel produced were tested with both antibody-free serums and serums known to contain antibodies specific to the appropriate surface antigens, and both positive and negative result controls were performed. The gels in the card products included in the test kit subject to the invention contain many types of gel swelling solutions. Swelling solutions with different pH, osmolite, and conductivity values were tested with existing gel matrices. The selection of the appropriate gel matrix and the appropriate swelling solution for this matrix was performed by evaluating the positive / negative results obtained from the interactions of HEK293 and / or CHO cells expressing erythrocyte antigen on the surface thereof with appropriate antibodies.

[0017] An erythrocyte-specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen subject to the invention comprises:

[0018] • ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens,

[0019] • HEK293 and / or CHO cell lines in which stable and overexpression of ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh- associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens will be performed,

[0020] • Complementary DNAs (cDNAs) of ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens for use as templates,

[0021] • agarose gel for performing quality control of polymerase chain reaction (PCR / PCR) products (insert),

[0022] • restriction enzymes for performing the task of truncation of the pLenti-lll-HA vector of the resulting PCR products,

[0023] • T4 ligase enzyme for the ligation of the digested PCR products obtained into the digested pLenti-l I l-HA vector,

[0024] • an isolation kit for plasmid isolation of inoculated colonies after incubation,

[0025] • restriction enzymes with which the isolated plasmids were treated to clarify whether the correct clones were obtained,

[0026] • QuickChange II Site Targeted Mutation kit for use in the creation of plasmids by mutagenic whole plasmid PCR and for obtaining antigens expressed as a result of one or more nucleotide substitutions,

[0027] • Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) for resuspending and flask seeding of HEK293 and / or F-12K medium supplemented with FBS for resuspending and flask seeding of CHO,

[0028] • carbon dioxide (CO2) for use in incubating cells,

[0029] • trypsin-ethylene diamine tetraacetic acid (EDTA) solution for use in removing cells from the surface to which they attach,

[0030] • medium for use in the inhibition of trypsin-EDTA activity,

[0031] • dimethyl sulfoxide (DMSO) to be used by being added to the medium in cells to be stocked in a liquid nitrogen tank,

[0032] • trypan blue for cell counting and cell density determination,

[0033] • psPAX2 (Addgene) and pCMV-VSV-G (Addgene), the packaging vectors with which the pLenti-ll l-HA vector will be added to the medium of the HEK293 cell line,

[0034] • polybrene (hexadimethrin bromide) for use in performing transduction into cells,

[0035] • a pipette for homogeneous dispersion of lentiviral particles after bringing them to room temperature and adding polybrene,

[0036] • a suspension of lentiviral particles for use as a mixture in which lentiviruses used to carry genetic material (DNA or RNA) into cells are suspended in liquid,

[0037] • serum dilution for use in suspending HEK293 and / or CHO cells, • Dulbecco's Modified Eagle's Medium (DMEM) supplemented with FBS for use to resuspend and seed the mentioned cells after collection of trypsin-EDTA removed cells by centrifugation,

[0038] • 2.5%, 1%, 0.5% and finally 0.1% FBS to acclimatize HEK293 cells to low serum medium and / or 2.5%, 1%, 0.6% and finally FBS-free (serum-free) medium to acclimatize CHO cells to low serum medium.

[0039] In an embodiment of the invention, an erythrocyte-specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen subject to the invention comprises:

[0040] • ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens,

[0041] • HEK293 and / or CHO cell lines in which stable and overexpression of ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh- associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens will be performed,

[0042] • Complementary DNAs (cDNAs) of ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens for use as templates,

[0043] • agarose gel for performing quality control of polymerase chain reaction (PCR / PCR) products (insert),

[0044] • restriction enzymes for performing the task of truncation of the pLenti-lll-HA vector of the resulting PCR products,

[0045] • T4 ligase enzyme for the ligation of the digested PCR products obtained into the digested pLenti-l I l-HA vector,

[0046] • an isolation kit for plasmid isolation of inoculated colonies after incubation, • restriction enzymes with which the isolated plasmids were treated to clarify whether the correct clones were obtained,

[0047] • QuickChange II Site Targeted Mutation kit for use in the creation of plasmids by mutagenic whole plasmid PCR and for obtaining antigens expressed as a result of one or more nucleotide substitutions,

[0048] • Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) for resuspending and flask seeding of HEK293 and / or F-12K medium supplemented with FBS for resuspending and flask seeding of CHO,

[0049] • carbon dioxide (CO2) for use in incubating cells,

[0050] • trypsin-ethylene diamine tetraacetic acid (EDTA) solution for use in removing cells from the surface to which they attach,

[0051] • medium for use in the inhibition of trypsin-EDTA activity,

[0052] • dimethyl sulfoxide (DMSO) to be used by being added to the medium in cells to be stocked in a liquid nitrogen tank,

[0053] • trypan blue for cell counting and cell density determination,

[0054] • psPAX2 (Addgene) and pCMV-VSV-G (Addgene), the packaging vectors with which the pLenti-ll l-HA vector will be added to the medium of the HEK293 cell line at 90% intensity,

[0055] • polybrene (hexadimethrin bromide) for use in performing transduction into cells at 80% intensity,

[0056] • a pipette for homogeneous dispersion of lentiviral particles stored at -80°C after bringing them to room temperature and adding polybrene,

[0057] • a suspension of lentiviral particles for use as a mixture in which lentiviruses used to carry genetic material (DNA or RNA) into cells are suspended in liquid,

[0058] • serum dilution for use in suspending HEK293 and / or CHO cells,

[0059] • Dulbecco's Modified Eagle's Medium (DMEM) supplemented with FBS for use to resuspend and seed the mentioned cells after collection of trypsin-EDTA removed cells by centrifugation,

[0060] • 2.5%, 1%, 0.5% and finally 0.1% FBS to acclimatize HEK293 cells to low serum medium and / or 2.5%, 1%, 0.6% and finally FBS-free (serum-free) medium to acclimatize CHO cells to low serum medium.

[0061] A preparation method for an erythrocyte-specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen subject to the invention comprises the process steps of: i. cloning ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens into pLenti-lll-HA (lentiviral) vector and performing stable and overexpression in HEK293 and / or CHO cell lines, ii. using complementary DNAs (cDNAs) of these antigens as templates and performing amplification of target genes with PrimeStar DNA polymerase, iii. performing the quality control (yield, specificity) of the polymerase chain reaction (PCR / PCR) products (insert) obtained on agarose gel and digesting the PCR products obtained using the relevant restriction enzymes for the pLenti-ll l-HA vector, and extracting the digested products from the agarose gel according to the manufacturer's protocol, iv. ligating the PCR products obtained as digested into the digested pLenti-lll-HA vector with T4 ligase enzyme, v. heat shock transforming the ligation products into competent E. coli DH5alpha bacteria, vi. plating agar medium containing the relevant resistance antibiotic, performing incubation, and observing colonies, vii. screening the colonies obtained and firstly finding the colonies containing the targeted plasmid by colony PCR and checking the PCR products obtained in agarose gel and inoculating the positive colonies into liquid medium so that there are at least two colonies for each target clone and ensuring plasmid isolation, viii. incubating inoculated colonies on a shaker followed by plasmid isolation using a mini isolation kit, ix. treating the isolated plasmids with the restriction enzymes with which the cloning was performed and clarifying whether the correct clones were obtained, x. checking on agarose gel that the relevant gene transferred into the vector is obtained at the same size as a result of enzyme digestion. xi. using the QuickChange II Site Targeted Mutation kit with the primers obtained, creating plasmids by mutagenic whole plasmid PCR and obtaining antigens expressed as a result of one or more nucleotide changes, xii. thawing HEK293 and / or CHO frozen cells and mixing with 9 ml of medium and centrifuging, xiii. resuspending the pellets obtained with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) for HEK293 and F-12K medium supplemented with FBS for CHO and seeding into flasks, xiv. incubating the cells under CO2 and harvesting them before they reach 90% density, xv. carefully removing the medium from the environment and sub-culturing the proliferated cells after the initial culture, xvi. removing cells from their attachment surface by exposure to trypsin-ethylene diamine tetraacetic acid (EDTA) solution, xvii. adding medium on top of the cells and stopping the Trypsin-EDTA activity, xviii. adding dimethyl sulfoxide (DMSO) to the medium for cells to be stocked in a liquid nitrogen tank, xix. cell counting with trypan blue and determining cell density, xx. using as many cells as needed by diluting or storing, xxi. performing lentiviral packaging for transduction and performing antigen expression of the resulting pLenti-l I l-HA vectors in HEK293 and / or CHO cells, xxii. adding the pLenti-ll l-HA vector together with the packaging vectors psPAX2 (Addgene) and pCMV-VSV-G (Addgene) into HEK293 cell line medium with 90% density in a petri dish followed by replacement of the medium with fresh medium, xxiii. collecting the first virus in the first 24 hours and collecting the second virus in the 48th hour following the medium change, xxiv. transferring the resulting virus suspension in tubes and storing, xxv. introducing lentiviral packages to HEK293 cells and / or CHO cells in separate cultures and performing desired erythrocyte surface antigen expression, xxvi. transducing with polybrene (hexadimethrin bromide) into cells in a petri dish, xxvii. bringing the stored lentiviral particles to room temperature and ensuring homogeneous dispersion using a pipette by adding polybrene, xxviii. removing medium from the cell and introducing lentiviral particle suspension, xxix. refreshing medium after transduction, xxx. eliminating cells that have not received the virus particle and, following the change of medium, initiating antibiotic selection in the cell and performing permanent expression of the transduced plasmids in the cell, xxxi. obtaining a polyclonal population of cells that stably express the desired plasmid at the end of selection, xxxii. obtaining a monoclonal cell line from the resulting polyclonal cell line by diluting the cells using the ‘limiting dilution’ Addgene protocol and calculating the dilution so that 0.5 cells fall per well, and then seeding the cells into a Petri dish so that a single cell falls into each well xxxiii. growing the cells in the cell line and analyzing the expression levels and selecting cells with no expression or expression levels below 10%, xxxiv. verifying whether the monoclonal cell lines obtained express the desired protein by protein immunoblot (Western Blot) method, xxxv. easy culturing of adherent cells and enabling genetic manipulations to be easily performed while preserving this characteristic, xxxvi. suspending HEK293 and / or CHO cells by serum dilution, xxxvii. collecting the cells removed by trypsin-EDTA by centrifugation and seeding by resuspending in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with FBS, xxxviii. growing the seeded cells until maximum cell density is reached, xxxix. examining the viability of the cells reaching the maximum density by staining with trypan blue and repeating the steps (xxxvii), (xxxviii), and (xxxix), each time using a lower concentration of FBS, xL acclimatising the cells to the low serum medium with 2.5%, 1%, 0.5% and finally 0.1% FBS additions respectively for HEK293 cells and / or using 2.5%, 1%, 0.6% and finally FBS-free medium respectively for CHO cells, and when switching to serum-free medium for CHO cells, supplementing the medium with pluronic additive and if cell viability drops below 80% at these stages, continuing after repeating the adaptation process by returning to the upper serum level, xli. growing cells by incubating cells in CO2in a shaking incubator after obtaining suspended cells.

[0062] In an embodiment of the invention, a preparation method of an erythrocyte-specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen subject to the invention comprises the process steps of: i. cloning ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens into pLenti-lll-HA (lentiviral) vector and performing stable and overexpression in HEK293 and / or CHO cell lines, ii. using complementary DNAs (cDNAs) of these antigens as templates and performing amplification of target genes with PrimeStar DNA polymerase, iii. performing the quality control (yield, specificity) of the polymerase chain reaction (PCR / PCR) products (insert) obtained on agarose gel and digesting the PCR products obtained using the relevant restriction enzymes for the pLenti-ll l-HA vector, and extracting the digested products from the agarose gel according to the manufacturer's protocol, iv. ligating the PCR products obtained as digested into the digested pLenti-lll-HA vector with T4 ligase enzyme, v. heat shock transforming the ligation products into competent E. coli DH5alpha bacteria, vi. plating agar medium containing the relevant resistance antibiotic, performing incubation at 37°C for a minimum of 16 hours, and observing colonies, vii. screening the colonies obtained and firstly finding the colonies containing the targeted plasmid by colony PCR and checking the PCR products obtained in agarose gel and inoculating the positive colonies into liquid medium so that there are at least two colonies for each target clone and ensuring plasmid isolation, viii. incubating inoculated colonies on a shaker at 37°C for a minimum of 16 hours followed by plasmid isolation using a mini isolation kit, ix. treating the isolated plasmids with the restriction enzymes with which the cloning was performed and clarifying whether the correct clones were obtained, x. checking on agarose gel that the relevant gene transferred into the vector is obtained at the same size as a result of enzyme digestion. xi. using the QuickChange II Site Targeted Mutation kit with the primers obtained, creating plasmids by mutagenic whole plasmid PCR and obtaining antigens expressed as a result of one or more nucleotide changes, xii. thawing HEK293 and / or CHO cells at 37°C for 2-4 minutes and mixing with 9 ml of medium and centrifuging at 125x g for 5-6 minutes, xiii. resuspending the pellets obtained with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for HEK293 and F- 12K medium supplemented with 10% FBS for CHO and seeding into 75 cm2flasks, xiv. incubating the cells under 5% CO2 and harvesting them before they reach 90% density, xv. carefully removing the medium from the environment and sub-culturing the proliferated cells after the initial culture, xvi. removing cells from their attachment surface by exposure to 0.25% trypsin-0.5 mM ethylene diamine tetraacetic acid (EDTA) solution, xvii. adding medium on top of the cells and stopping the Trypsin-EDTA activity, xviii. adding 5% dimethyl sulfoxide (DMSO) to the medium for cells to be stocked in a liquid nitrogen tank, xix. cell counting with trypan blue and determining cell density, xx. using as many cells as needed by diluting or storing, xxi. performing lentiviral packaging for transduction and performing antigen expression of the resulting pLenti-l I l-HA vectors in HEK293 and / or CHO cells, xxii. adding the pLenti-ll l-HA vector together with the packaging vectors psPAX2 (Addgene) and pCMV-VSV-G (Addgene) into HEK293 cell line medium with 90% density in a 10 cm petri dish followed by replacement of the medium with fresh medium after 16 hours, xxiii. collecting the first virus in the first 24 hours and collecting the second virus in the 48th hour following the medium change, xxiv. transferring the virus suspension obtained into 2 mL tubes and storing at -80°C, xxv. introducing lentiviral packages to HEK293 cells and / or CHO cells in separate cultures and performing desired erythrocyte surface antigen expression, xxvi. transducing with polybrene (hexadimethrin bromide) into cells at 80% intensity in a 6-well petri dish, xxvii. bringing the lentiviral particles stored at -80°C to room temperature and ensuring homogeneous dispersion using a pipette by adding polybrene, xxviii. removing medium from the cell and introducing lentiviral particle suspension, xxix. refreshing medium 16 hours after transduction, xxx. eliminating cells that have not received the virus particle and, following the change of medium, initiating antibiotic selection in the cell and performing permanent expression of the transduced plasmids in the cell, xxxi. obtaining a polyclonal population of cells that stably express the desired plasmid after a selection of 1 -2 weeks, xxxii. obtaining a monoclonal cell line from the resulting polyclonal cell line by diluting the cells using the ‘limiting dilution’ Addgene protocol and calculating the dilution so that 0.5 cells fall per well, and then seeding the cells into a 96-well petri dish so that a single cell falls into each well xxxiii. growing the cells in the cell line and analyzing the expression levels and selecting cells with no expression or expression levels below 10%, xxxiv. verifying whether the monoclonal cell lines obtained express the desired protein by protein immunoblot (Western Blot) method, xxxv. easy culturing of adherent cells and enabling genetic manipulations to be easily performed while preserving this characteristic, xxxvi. suspending HEK293 and / or CHO cells by serum dilution, xxxvii. collecting the cells removed by trypsin-EDTA by centrifugation and seeding by resuspending in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% FBS, xxxviii. growing the seeded cells until maximum cell density is reached, xxxix. examining the viability of the cells reaching the maximum density by staining with trypan blue and repeating the steps (xxxvii), (xxxviii), and (xxxix), each time using a lower concentration of FBS, xl. acclimatising the cells to the low serum medium with 2.5%, 1%, 0.5% and finally 0.1% FBS additions respectively for HEK293 cells and / or using 2.5%, 1%, 0.6% and finally FBS-free (serum-free) medium respectively for CHO cells, and when switching to serum-free medium for CHO cells, supplementing the medium with pluronic additive and if cell viability drops below 80% at these stages, continuing after repeating the adaptation process by returning to the upper serum level, xli. growing cells by incubating cells in 5% CO2in a shaking incubator (50-80 rpm) after obtaining suspended cells.

[0063] The expression "125x g" mentioned in the method refers to the speed of a centrifuge in terms of gravitational force, and such an expression is generally used for high-speed and powerful centrifuges.

[0064] While preparing the erythrocyte-specific antibody screening test kit, the targeted antigens will be described using the HEK293 cell line, which can rapidly proliferate in cell culture and can be easily transfected. While preparing the erythrocyte-specific antibody screening test kit subject to the invention, genetic modifications are made on cell lines in cell culture in vitro and cells expressing a single antigen from target erythrocyte (RBC) antigens are obtained. By way of example, in the state of the art, in the study conducted with cell panels containing both Kell blood group system antigens K and k antigens and Duffy blood group antigens Fya and Fyb antigens on the erythrocyte (RBC) surface, it is impossible to distinguish whether the positive reaction is due to the presence of Kell antibodies or Duffy antibodies in the patient serum / plasma. Since only K or k antigens belonging to the Kell blood group system antigens will be carried at one time while preparing the erythrocyte-specific antibody screening test kit subject to the invention, it will be possible to accurately determine the presence of antibodies in the patient serum.

[0065] In the test kit subject to the invention, results can be obtained with simple test techniques such as gel centrifugation method or slide method without the need for any extra device etc. system. In the test kit subject to the invention, the patient sample is studied with an antibody screening-identification cell to be produced using a coombs card containing a dextran-derived gel. After incubation for 10-15 minutes at 35-37 C, the results are evaluated by their severity from +- to 4 positivity.

[0066] HEK293 cell line and / or CHO cells are used in the preparation of the test kit subject to the invention and RBC antigens are not included in the cell membranes of CHO cells. Table 1 presents erythrocyte antigen expression profiles in HEK293 cells of human origin.

[0067] Table 1. RBC Antigen Expression Profile of the HEK293 Cell Line.

[0068] While preparing the erythrocyte-specific antibody screening test kit, the targeted antigens will be described using the HEK293 cell line, which can rapidly proliferate in cell culture and can be easily transfected. Said antigen systems are ABO, MNS, P1 PK, 5 Rh (RH), Lutheran (LU), KEII (KEL), Lewis (LE), Duffy (FY), Kidd (JK), Diego (DI), Yt (YT), Xg (XG), Scianna (SC), Dombrock (DO), Colton (CO), Landsteiner-Wiener (LW), Chido / Rodgers (CH / RG), H, Kx (XK), Gerbich (GE), Cromer (CROM), Knops (KN), Indian (IN), Ok (OK), Raph (RAPH), JohnMiltonHagen (JMH), I, Globoside (GLOB), Gill (GIL), Rh-associated glycoprotein (RHAG), FORS, JR, LAN, Vel (VEL), CD59, 0 Augustine (AUG), Kanno (KANNO), SID, CTL2, PEL, MAM, EMM, ABCC1 , Er (ER), and CD36. Detailed information on these antigen systems is presented in Table 2.

[0069] Table 2. Details of the target antigens described in the preparation of the test kit subject to the invention.

[0070]

[0071] In the present art, blood transfusions can be performed if the results are suitable between the recipient and the donor after the suitability tests performed before blood transfusion in the Immunohematology laboratories in the Blood Centers. After ABO 5 blood grouping, the most important tests are crossmatch and antibody screening / identification tests. Antibody identification tests contain a certain concentration of human blood of known phenotype. The patient's blood (plasma) is tested with these cells to determine the presence and / or absence of antibodies. In the specific antibody screening test kit subject to the invention, the antibody identification 0 cells will be cells specifically designed with genetic modification; in other words, they will be cells containing only one phenotype. In this way, a cell that is positive in a patient sample will be reported as positive for that phenotype and a cell that is negative will be reported as negative for that phenotype. The ultimate objective of the test kit subject to the invention is that the cells obtained specific to each antigen can be used 5 instead of 2, 3 and 4-cell screening panels and 11 -cell identification panels. The antibody screening tests performed with erythrocyte cell suspensions used in the state of the art without reaching a definitive conclusion based on the antigenic panel are insufficient for the precise and accurate detection of the patient’s antibodies. These tests are performed using commercially available 2-, 3-, 4-screening and 11 - 0 identification erythrocyte cell suspension panels. In the test kit subject to the invention, besides the screening of serum antibodies, the production of antigens targeting the screening of other antibodies (bacterial, viral infections, etc.) in case of need, provides the clearest results in antibody screening. Industrial Applicability of the Invention

[0072] The invention relates to an erythrocyte specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen (RhD and RhCE as 2 different antigens derived from RBC and 45 or more antigens in total including some variants of these two antigens) and a preparation method of this test kit, and is industrially applicable.

[0073] The invention is not limited to the above descriptions and the person skilled in the art can readily present other different embodiments of the invention. These should be considered within the protection scope of the invention claimed by the claims.

[0074] References

[0075] [1] Antikor Testi Nedir ve Kimlere Yapilabilir? - Medicana. (n.d.). https: / / www.medicana.com.tr / saglik-rehberi-detay / 10398 / antikor-testi-nedir-ve kimlere- yapilabilir

Claims

CLAIMS1 . An erythrocyte-specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen, characterized in that it comprises:• ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens,• HEK293 and / or CHO cell lines in which stable and overexpression of ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens will be performed,• Complementary DNAs (cDNAs) of ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens for use as templates,• agarose gel for performing quality control of polymerase chain reaction (PCR / PCR) products (insert),• restriction enzymes for performing the task of truncation of the pLenti-l II- HA vector of the resulting PCR products,• T4 ligase enzyme for the ligation of the digested PCR products obtained into the digested pLenti-l I l-HA vector,• an isolation kit for plasmid isolation of inoculated colonies after incubation,• restriction enzymes with which the isolated plasmids were treated to clarify whether the correct clones were obtained,• QuickChange II Site Targeted Mutation kit for use in the creation of plasmids by mutagenic whole plasmid PCR and for obtaining antigens expressed as a result of one or more nucleotide substitutions,• Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) for resuspending and flask seeding of HEK293 and / or F-12K medium supplemented with FBS for resuspending and flask seeding of CHO,• carbon dioxide (CO2) for use in incubating cells,• trypsin-ethylene diamine tetraacetic acid (EDTA) solution for use in removing cells from the surface to which they attach,• medium for use in the inhibition of trypsin-EDTA activity,• dimethyl sulfoxide (DMSO) to be used by being added to the medium in cells to be stocked in a liquid nitrogen tank,• trypan blue for cell counting and cell density determination,• psPAX2 (Addgene) and pCMV-VSV-G (Addgene), the packaging vectors with which the pLenti-l ll-HA vector will be added to the medium of the HEK293 cell line,• polybrene (hexadimethrin bromide) for use in performing transduction into cells,• a pipette for homogeneous dispersion of lentiviral particles after bringing them to room temperature and adding polybrene,• a suspension of lentiviral particles for use as a mixture in which lentiviruses used to carry genetic material (DNA or RNA) into cells are suspended in liquid,• serum dilution for use in suspending HEK293 and / or CHO cells,• Dulbecco's Modified Eagle's Medium (DMEM) supplemented with FBS for use to resuspend and seed the mentioned cells after collection of trypsin-EDTA removed cells by centrifugation,• 2.5%, 1%, 0.5% and finally 0.1% FBS to acclimatize HEK293 cells to low serum medium and / or 2.5%, 1%, 0.6% and finally FBS-free (serum-free) medium to acclimatize CHO cells to low serum medium.

2. An erythrocyte-specific antibody screening test kit according to claim 1 , characterized in that it comprises:• ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens,• HEK293 and / or CHO cell lines in which stable and overexpression of ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens will be performed,• Complementary DNAs (cDNAs) of ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens for use as templates,• agarose gel for performing quality control of polymerase chain reaction (PCR / PCR) products (insert),• restriction enzymes for performing the task of truncation of the pLenti-l II- HA vector of the resulting PCR products,• T4 ligase enzyme for the ligation of the digested PCR products obtained into the digested pLenti-l I l-HA vector,• an isolation kit for plasmid isolation of inoculated colonies after incubation,• restriction enzymes with which the isolated plasmids were treated to clarify whether the correct clones were obtained,• QuickChange II Site Targeted Mutation kit for use in the creation of plasmids by mutagenic whole plasmid PCR and for obtaining antigens expressed as a result of one or more nucleotide substitutions,• Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) for resuspending and flask seeding of HEK293and / or F-12K medium supplemented with FBS for resuspending and flask seeding of CHO,• carbon dioxide (CO2) for use in incubating cells,• trypsin-ethylene diamine tetraacetic acid (EDTA) solution for use in removing cells from the surface to which they attach,• medium for use in the inhibition of trypsin-EDTA activity,• dimethyl sulfoxide (DMSO) to be used by being added to the medium in cells to be stocked in a liquid nitrogen tank,• trypan blue for cell counting and cell density determination,• psPAX2 (Addgene) and pCMV-VSV-G (Addgene), the packaging vectors with which the pLenti-l ll-HA vector will be added to the medium of the HEK293 cell line at 90% intensity,• polybrene (hexadimethrin bromide) for use in performing transduction into cells at 80% intensity,• a pipette for homogeneous dispersion of lentiviral particles stored at - 80°C after bringing them to room temperature and adding polybrene,• a suspension of lentiviral particles for use as a mixture in which lentiviruses used to carry genetic material (DNA or RNA) into cells are suspended in liquid,• serum dilution for use in suspending HEK293 and / or CHO cells,• Dulbecco's Modified Eagle's Medium (DMEM) supplemented with FBS for use to resuspend and seed the mentioned cells after collection of trypsin-EDTA removed cells by centrifugation,• 2.5%, 1%, 0.5% and finally 0.1% FBS to acclimatize HEK293 cells to low serum medium and / or 2.5%, 1%, 0.6% and finally FBS-free (serum-free) medium to acclimatize CHO cells to low serum medium.

3. A preparation method for the erythrocyte-specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen, characterized in that it comprises the process steps of: i. cloning ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM,ABCC1 , Er or CD36 antigens into pLenti-l ll-HA (lentiviral) vector and performing stable and overexpression in HEK293 and / or CHO cell lines, ii. using complementary DNAs (cDNAs) of these antigens as templates and performing amplification of target genes with PrimeStar DNA polymerase, iii. performing the quality control (yield, specificity) of the polymerase chain reaction (PCR / PCR) products (insert) obtained on agarose gel and digesting the PCR products obtained using the relevant restriction enzymes for the pLenti-l ll-HA vector, and extracting the digested products from the agarose gel according to the manufacturer's protocol, iv. ligating the PCR products obtained as digested into the digested pLenti- lll-HA vector with T4 ligase enzyme, v. heat shock transforming the ligation products into competent E. coli DH5alpha bacteria, vi. plating agar medium containing the relevant resistance antibiotic, performing incubation, and observing colonies, vii. screening the colonies obtained and firstly finding the colonies containing the targeted plasmid by colony PCR and checking the PCR products obtained in agarose gel and inoculating the positive colonies into liquid medium so that there are at least two colonies for each target clone and ensuring plasmid isolation, viii. incubating inoculated colonies on a shaker followed by plasmid isolation using a mini isolation kit, ix. treating the isolated plasmids with the restriction enzymes with which the cloning was performed and clarifying whether the correct clones were obtained, x. checking on agarose gel that the relevant gene transferred into the vector is obtained at the same size as a result of enzyme digestion. xi. using the QuickChange II Site Targeted Mutation kit with the primers obtained, creating plasmids by mutagenic whole plasmid PCR and obtaining antigens expressed as a result of one or more nucleotide changes, xii. thawing HEK293 and / or CHO frozen cells and mixing with 9 ml of medium and centrifuging,xiii. resuspending the pellets obtained with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) for HEK293 and F-12K medium supplemented with FBS for CHO and seeding into flasks, xiv. incubating the cells under CO2 and harvesting them before they reach 90% density, xv. carefully removing the medium from the environment and sub-culturing the proliferated cells after the initial culture, xvi. removing cells from their attachment surface by exposure to trypsinethylene diamine tetraacetic acid (EDTA) solution, xvii. adding medium on top of the cells and stopping the Trypsin-EDTA activity, xviii. adding dimethyl sulfoxide (DMSO) to the medium for cells to be stocked in a liquid nitrogen tank, xix. cell counting with trypan blue and determining cell density, xx. using as many cells as needed by diluting or storing, xxi. performing lentiviral packaging for transduction and performing antigen expression of the resulting pLenti-l I l-HA vectors in HEK293 and / or CHO cells, xxii. adding the pLenti-ll l-HA vector together with the packaging vectors psPAX2 (Addgene) and pCMV-VSV-G (Addgene) into HEK293 cell line medium with 90% density in a petri dish followed by replacement of the medium with fresh medium, xxiii. collecting the first virus in the first 24 hours and collecting the second virus in the 48th hour following the medium change, xxiv. transferring the resulting virus suspension in tubes and storing, xxv. introducing lentiviral packages to HEK293 cells and / or CHO cells in separate cultures and performing desired erythrocyte surface antigen expression, xxvi. transducing with polybrene (hexadimethrin bromide) into cells in a petri dish, xxvii. bringing the stored lentiviral particles to room temperature and ensuring homogeneous dispersion using a pipette by adding polybrene, xxviii. removing medium from the cell and introducing lentiviral particle suspension,xxix. refreshing medium after transduction, xxx. eliminating cells that have not received the virus particle and, following the change of medium, initiating antibiotic selection in the cell and performing permanent expression of the transduced plasmids in the cell, xxxi. obtaining a polyclonal population of cells that stably express the desired plasmid at the end of selection, xxxii. obtaining a monoclonal cell line from the resulting polyclonal cell line by diluting the cells using the ‘limiting dilution’ Addgene protocol and calculating the dilution so that 0.5 cells fall per well, and then seeding the cells into a Petri dish so that a single cell falls into each well xxxiii. growing the cells in the cell line and analyzing the expression levels and selecting cells with no expression or expression levels below 10%, xxxiv. verifying whether the monoclonal cell lines obtained express the desired protein by protein immunoblot (Western Blot) method, xxxv. easy culturing of adherent cells and enabling genetic manipulations to be easily performed while preserving this characteristic, xxxvi. suspending HEK293 and / or CHO cells by serum dilution, xxxvii. collecting the cells removed by trypsin-EDTA by centrifugation and seeding by resuspending in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with FBS, xxxviii. growing the seeded cells until maximum cell density is reached, xxxix. examining the viability of the cells reaching the maximum density by staining with trypan blue and repeating the steps (xxxvii), (xxxviii), and (xxxix), each time using a lower concentration of FBS, xl. acclimatising the cells to the low serum medium with 2.5%, 1%, 0.5% and finally 0.1% FBS additions respectively for HEK293 cells and / or using 2.5%, 1%, 0.6% and finally FBS-free medium respectively for CHO cells, and when switching to serum-free medium for CHO cells, supplementing the medium with pluronic additive and if cell viability drops below 80% at these stages, continuing after repeating the adaptation process by returning to the upper serum level, xli. growing cells by incubating cells in CO2in a shaking incubator after obtaining suspended cells.

4. A preparation method according to claim 3, characterized in that it comprises the process steps of; i. cloning ABO, MNS, P1 PK, Rh, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, JohnMiltonHagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, SID, CTL2, PEL, MAM, EMM, ABCC1 , Er or CD36 antigens into pLenti-l I l-HA (lentiviral) vector and performing stable and overexpression in HEK293 and / or CHO cell lines, ii. using complementary DNAs (cDNAs) of these antigens as templates and performing amplification of target genes with PrimeStar DNA polymerase, iii. performing the quality control (yield, specificity) of the polymerase chain reaction (PCR / PCR) products (insert) obtained on agarose gel and digesting the PCR products obtained using the relevant restriction enzymes for the pLenti-l ll-HA vector, and extracting the digested products from the agarose gel according to the manufacturer's protocol, iv. ligating the PCR products obtained as digested into the digested pLenti-l I l-HA vector with T4 ligase enzyme, v. heat shock transforming the ligation products into competent E. coli DH5alpha bacteria, vi. plating agar medium containing the relevant resistance antibiotic, performing incubation at 37°C for a minimum of 16 hours, and observing colonies, vii. screening the colonies obtained and firstly finding the colonies containing the targeted plasmid by colony PCR and checking the PCR products obtained in agarose gel and inoculating the positive colonies into liquid medium so that there are at least two colonies for each target clone and ensuring plasmid isolation, viii. incubating inoculated colonies on a shaker at 37°C for a minimum of 16 hours followed by plasmid isolation using a mini isolation kit,ix. treating the isolated plasmids with the restriction enzymes with which the cloning was performed and clarifying whether the correct clones were obtained, x. checking on agarose gel that the relevant gene transferred into the vector is obtained at the same size as a result of enzyme digestion. xi. using the QuickChange II Site Targeted Mutation kit with the primers obtained, creating plasmids by mutagenic whole plasmid PCR and obtaining antigens expressed as a result of one or more nucleotide changes, xii. thawing HEK293 and / or CHO cells at 37°C for 2-4 minutes and mixing with 9 ml of medium and centrifuging at 125x g for 5-6 minutes, xiii. resuspending the pellets obtained with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for HEK293 and F-12K medium supplemented with 10% FBS for CHO and seeding into 75 cm2flasks, xiv. incubating the cells under 5% CO2and harvesting them before they reach 90% density, xv. carefully removing the medium from the environment and subculturing the proliferated cells after the initial culture, xvi. removing cells from their attachment surface by exposure to 0.25% trypsin-0.5 mM ethylene diamine tetraacetic acid (EDTA) solution, xvii. adding medium on top of the cells and stopping the Trypsin-EDTA activity, xviii. adding 5% dimethyl sulfoxide (DMSO) to the medium for cells to be stocked in a liquid nitrogen tank, xix. cell counting with trypan blue and determining cell density, xx. using as many cells as needed by diluting or storing, xxi. performing lentiviral packaging for transduction and performing antigen expression of the resulting pLenti-ll l-HA vectors in HEK293 and / or CHO cells, xxii. adding the pLenti-l I l-HA vector together with the packaging vectors psPAX2 (Addgene) and pCMV-VSV-G (Addgene) into HEK293 cell line medium with 90% density in a 10 cm petri dish followed by replacement of the medium with fresh medium after 16 hours,xxiii. collecting the first virus in the first 24 hours and collecting the second virus in the 48th hour following the medium change, xxiv. transferring the virus suspension obtained into 2 mL tubes and storing at -80°C, xxv. introducing lentiviral packages to HEK293 cells and / or CHO cells in separate cultures and performing desired erythrocyte surface antigen expression, xxvi. transducing with polybrene (hexadimethrin bromide) into cells at 80% intensity in a 6-well petri dish, xxvii. bringing the lentiviral particles stored at -80°C to room temperature and ensuring homogeneous dispersion using a pipette by adding polybrene, xxviii. removing medium from the cell and introducing lentiviral particle suspension, xxix. refreshing medium 16 hours after transduction, xxx. eliminating cells that have not received the virus particle and, following the change of medium, initiating antibiotic selection in the cell and performing permanent expression of the transduced plasmids in the cell, xxxi. obtaining a polyclonal population of cells that stably express the desired plasmid after a selection of 1 -2 weeks, xxxii. obtaining a monoclonal cell line from the resulting polyclonal cell line by diluting the cells using the ‘limiting dilution’ Addgene protocol and calculating the dilution so that 0.5 cells fall per well, and then seeding the cells into a 96-well petri dish so that a single cell falls into each well xxxiii. growing the cells in the cell line and analyzing the expression levels and selecting cells with no expression or expression levels below 10%, xxxiv. verifying whether the monoclonal cell lines obtained express the desired protein by protein immunoblot (Western Blot) method, xxxv. easy culturing of adherent cells and enabling genetic manipulations to be easily performed while preserving this characteristic, xxxvi. suspending HEK293 and / or CHO cells by serum dilution,xxxvii. collecting the cells removed by trypsin-EDTA by centrifugation and seeding by resuspending in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% FBS, xxxviii. growing the seeded cells until maximum cell density is reached, xxxix. examining the viability of the cells reaching the maximum density by staining with trypan blue and repeating the steps (xxxvii), (xxxviii), and (xxxix), each time using a lower concentration of FBS, xl. acclimatising the cells to the low serum medium with 2.5%, 1%, 0.5% and finally 0.1% FBS additions respectively for HEK293 cells and / or using 2.5%, 1%, 0.6% and finally FBS-free medium respectively for CHO cells, and when switching to serum-free medium for CHO cells, supplementing the medium with pluronic additive and if cell viability drops below 80% at these stages, continuing after repeating the adaptation process by returning to the upper serum level, xli. growing cells by incubating cells in 5% CO2 in a shaking incubator (50-80 rpm) after obtaining suspended cells.

5. An erythrocyte-specific antibody screening test kit expressing a single antigen from a target erythrocyte (RBC) antigen prepared by a method according to any one of claims 3 or 4.

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