Methods and device for determining Anti-blood group antibodies

Abstract

The invention relates to methods for determining an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample, comprising the following steps: a) providing a biological sample comprising erythrocytes, wherein the erythrocytes express a transport protein (CD38); and b) providing a polypeptide which is specific to CD38 and comprises an amino acid sequence; and c) contacting and incubating the biological sample with the polypeptide; and d) determining an anti-blood group antibody and / or an anti-CD38 antibody in the biological sample. The invention further relates to a device and a kit for determining an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample, and to an expression system for producing a polypeptide according to the invention.

Description

[0001] December 23, 2025

[0002] BAG Diagnostics GmbH G / BGD-006-WO 35423 Lieh Sew

[0003] Method and apparatus for determining anti-blood group antibodies

[0004] The invention relates to a method, a device and a kit for determining an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample according to the preamble of the independent claims.

[0005] A blood group describes the individual composition of structures on the outer membrane of erythrocytes in vertebrates within a blood group system. The surfaces of the outer membrane differ due to various glycolipids or proteins that act as antigens and can thus trigger an immune response. Blood groups are of particular importance in transfusion and transplantation medicine because there is a risk that the immune system of a patient receiving blood of an incompatible blood group will overreact. This overreaction of the immune system manifests as defense mechanisms that lead to blood clotting (agglutination) and can cause serious damage, even death. In transplantation medicine, this can lead, among other things, to rejection of the transplanted organ.The established human therapeutic CD38 antibodies, such as daratumumab and isatuximab, both human monoclonal immunoglobulin IgGk antibodies (mAbs) for the treatment of patients with multiple myeloma, systemic light chain amyloidosis, and / or monoclonal gammopathy, bind to two different epitopes of the transmembrane protein CD38, which is expressed, among other things, on malignant myeloma cells as well as on erythrocytes. A problem has arisen because blood plasma samples from patients treated with these therapeutic antibodies for multiple myeloma, systemic light chain amyloidosis, and / or monoclonal gammopathy consistently produce false-positive agglutination reactions in indirect antiglobulin tests. These are caused by an unwanted agglutination of red blood cells (RBCs, erythrocytes), which can occur even months after the last infusion.

[0006] It is therefore not surprising that state-of-the-art applications exist for neutralizing these therapeutic antibodies in indirect antiglobulin tests, enabling the continued reliable determination of patients' blood groups. However, these applications are problematic because they are expensive to manufacture, too nonspecific, too time-consuming, and also have a short shelf life.

[0007] Furthermore, it is known from the prior art to neutralize cross-reactions and / or interfering factors occurring in indirect antiglobulin tests using chemical and / or enzymatic methods. However, this is too nonspecific, so that in addition to CD38, certain blood group antigens on the surface of the erythrocytes are also destroyed, which are then no longer natively available for blood group determination or for screening for irregular blood group antibodies.

[0008] There is therefore a great need for a method, device, and kit for the simplified determination of anti-blood group antibodies on erythrocytes in the presence of interfering therapeutic anti-CD38 antibodies, particularly in vitro, with a further emphasis on cost-effectiveness. Furthermore, the determination should be reliable, simple, specific, and rapid, as well as user-friendly and achievable within standard user protocols. Another key requirement is that the determination can be reliably performed even in the presence of cross-reactions and / or interfering factors, such as interference signals in indirect anti-human globulin assays, and that the analyzed biological sample is readily available for further analyses, such as blood group determination or screening for irregular blood group antibodies.Another aspect is that the method and the device can be carried out practically without further instructions, and the interpretation is clear and unambiguous. The invention therefore aims to provide a method, a device, and a kit for determining anti-blood group antibodies and / or anti-CD38 antibodies in a biological sample, in order to avoid the aforementioned disadvantages of the prior art and to ensure high efficacy, specificity, and / or reliability while simultaneously ensuring simple, low-error, time-efficient, and cost-effective application.

[0009] This problem is solved in a surprisingly simple but effective way by a method, a device and a kit for determining an anti-blood group antibody and / or an anti-CD38 antibody according to the teaching of the independent claims.

[0010] According to the invention, an analytical, in vitro method for determining an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample is proposed, comprising the following steps: a) providing a biological sample comprising erythrocytes, in particular an erythrocyte suspension, wherein the erythrocytes express a transport protein (CD38); and

[0011] b) Providing at least one polypeptide, in particular a single-domain polypeptide, which is specific for CD38 and comprises an amino acid sequence, wherein the amino acid sequence is selected from the group comprising:

[0012] i) an amino acid sequence according to SEQ ID No. 1 to SEQ ID No. 58, which codes for an amino acid molecule; and

[0013] ii) an amino acid sequence encoding an amino acid molecule that is at least 50% identical to the amino acid molecule encoded by the amino acid sequence from i); and

[0014] iii) an amino acid sequence for a fragment of an amino acid molecule from i) or ii), wherein the fragment encodes a polypeptide; and

[0015] c) Bringing the biological sample into contact with the polypeptide and incubating it; and

[0016] d) Determination of an anti-blood group antibody and / or an anti-CD38 antibody in the biological sample.

[0017] The basic idea of ​​the present invention is to provide a method for the simplified determination of anti-blood group antibodies on erythrocytes in a biological sample, which is also reliable in the presence of interfering therapeutic anti-CD38 antibodies, whereby their presence or absence is also to be determined. Within the scope of the invention, it has been recognized that cross-reactions and / or interfering factors can be reliably neutralized by the use of polypeptides with specific properties.

[0018] The term "blood group" generally refers to one of many groups / types into which the blood of a human or animal patient can be categorized based on the presence or absence of specific antigens, preferably produced by the same gene, on the surface of blood cells. Within the scope of the invention, the term "blood group" refers to the combination of surface antigens of the erythrocytes and / or the erythrocyte suspension, i.e., the red blood cells (RBCs), of a human or animal patient. Within the scope of the invention, the highly immunogenic Kell blood group system, particularly with the two principal antigens Kell (K; Kl) and Cellano (k; K2), the ABO blood group system, and / or the Rhesus blood group system (Rh) are preferred. Various blood group antigens are conceivable within the scope of the invention, as described elsewhere.

[0019] The term "anti-blood group antibody" is known to those skilled in the art and refers to antibodies formed against the antigens of a blood group, in particular those directed against the antigens on the surface of blood cells, preferably against the surface antigens of erythrocytes and / or the erythrocyte suspension, i.e., red blood cells (RBCs). It is understandable that a patient, i.e., a human or an animal, with blood group Kell-negative (k,k) forms antibodies against Kell (K, K) and that a patient with blood group Kell-positive (K, K) forms antibodies against Cellano. Various blood group antigens are conceivable within the scope of the invention, as described elsewhere.

[0020] In the first step of the process according to the invention, it is necessary to provide a biological sample comprising erythrocytes, wherein the erythrocytes express a transport protein (CD38). The term "biological sample" refers to a material of human and / or animal origin which, according to the invention, comprises erythrocytes, in particular an erythrocyte suspension. It is conceivable that the biological sample is present as a suspension, for example in a concentration of at least 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1%, 1.05%, 1.10%, 1.15%, 1.20%, 1.25%, 1.30%, 1.35%, 1.40%, 1.45%, 1.50%, 1.55%, 1.60%, 1.65%, 1.70%, 1.75%, 1.80%, 1.85%, 1.90%, 1.95%, 1.96%, 1.97%, 1.98%, 1.99%, or 2%.It is conceivable that the blood group antigens are predetermined on the cell membrane of the erythrocytes and / or the erythrocyte suspension, such as panel cells or donor test cells.

[0021] Such previously described biological samples are known to those skilled in the art, such as erythrocyte-containing samples from, among other things, saliva, urine, blood, stool, cellular tissue, organ punctures, organs, parts of organs, or similar samples. It is also known to those skilled in the art that erythrocytes express, among other things, CD38, which is a multifunctional transmembrane protein and functions as a lymphocyte receptor. It is of secondary importance whether the biological sample is treated or untreated. The term "treated" means that the biological sample originates from a patient who, due to an existing disease such as multiple myeloma, systemic light chain amyloidosis, or monoclonal gammopathy, has been treated with a therapeutic anti-CD38 antibody, wherein the anti-CD38 antibody is specific for CD38.Such therapeutic anti-CD38 antibodies are known to a specialist, such as, but by no means exclusively, daratumumab, felzartamab, mezagitamab, erzotabart, CM313 and / or isatuximab.

[0022] In the next step of the process according to the invention, it is necessary to provide at least one polypeptide, in particular a single-domain polypeptide, which is specific for CD38 and has an amino acid sequence. Preferably, the polypeptide is specific for at least one epitope of the CD38 surface antigen of the erythrocyte and / or the erythrocyte suspension, in particular the erythrocyte membrane. More preferably, the polypeptide is specific for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the aforementioned epitope, as well as for a combination of certain epitopes of the CD38 surface antigen of the erythrocyte. Furthermore, the polypeptide forms at least one paratope with high specificity for the CD38 antigen. Preferably, the polypeptide does not have a human and / or animal Fc region, which is present, for example, in therapeutic IgG antibodies.This is important because otherwise interference with the procedure could occur, particularly when determining an anti-blood group antibody in the biological sample. The single-domain polypeptide is preferably monospecific, bi-, tri-, or multispecific and / or mono-, bi-, tri-, or multivalent. Preferably, the polypeptide is a monovalent polypeptide that binds to at least one specific epitope of the CD38 antigen.

[0023] Preferably, the polypeptide is a monoclonal antibody, a polyclonal antibody, or a combination thereof. Within the scope of the invention, it has been found that the provided polypeptide partially or completely blocks and / or inhibits the binding sites or the binding of therapeutic anti-CD38 antibodies, such as daratumumab, isatuximab, mezagitamab, erzotabart, CM313, and / or felzartamab, on erythrocytes, particularly on the surface of a membrane-bound CD38, and thus neutralizes them. It is conceivable that the polypeptide and the therapeutic anti-CD38 antibody cover the same epitope region(s) of the CD38 epitope and / or that the epitope regions overlap.

[0024] Preferably, the efficacy of blocking, inhibiting, and / or neutralizing is at least 50%, more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.99%, or 100%. It is understood that an efficacy of at least 70% for blocking, inhibiting, and / or neutralizing is considered significant, and that an efficacy of at least 95% for blocking, inhibiting, and / or neutralizing is considered significant. It is known to those skilled in the art that a reduction of 100% is in the rarest of cases, however this can certainly be achieved by means of the inventive method or the inventive polypeptide.Therefore, within the scope of the invention, it is understandable that the polypeptide is referred to as a “blocker” and / or that the method is also or alternatively a method for blocking, inhibiting and / or neutralizing an anti-CD38 antibody in a biological sample.

[0025] The term "block" means that the CD38 antigen of the erythrocytes is occupied in such a way that the therapeutic anti-CD38 antibody is prevented from binding to them. Within the scope of the invention, it has been discovered that the polypeptide occupies the CD38 antigen of the erythrocytes. This blocking can occur partially or completely.

[0026] The term "inhibit" means that the binding of the therapeutic anti-CD38 antibody to the CD38 antigen of the erythrocytes is prevented and / or the possibility of the antibody binding to the antigen is reduced. This can occur, for example, if the polypeptide competes with the therapeutic anti-CD38 antibody. This inhibition can be partial or complete.

[0027] To a specialist, it is clear that the terms "polypeptide," "peptide," "amino acids," and "protein" are used interchangeably to describe a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Thus, amino acids include, but are by no means limited to, naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified.Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonylmethane. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of an amino acid, but function similarly to a naturally occurring amino acid. Polypeptides can function as storage molecules, transport and / or signaling molecules, structural molecules, protective and / or defensive molecules, and / or metabolically active molecules, performing various functions within and / or outside a cell or organism.Polypeptides can exist as simple linear polypeptides or as folded structural proteins.

[0028] In the process according to the invention, at least one amino acid molecule, one polypeptide, and / or a fragment thereof is provided; preferably, the polypeptide can also encode 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid molecules, polypeptides, and / or fragments thereof. It is conceivable that these polypeptides represent different clones of a polypeptide which bind specifically to certain epitopes of the CD38 antigen.

[0029] It is conceivable that an amino acid sequence, as described in SEQ ID No. 1 to SEQ ID No. 58 (see Table 1), could code for a naturally occurring or synthetic amino acid molecule.

[0030] The amino acid sequence is preferred as in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57 or SEQ ID No. 58 described.Furthermore, an amino acid sequence is conceivable that codes for an amino acid molecule, a polypeptide, and / or a fragment thereof, which is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the previously mentioned amino acid molecule. The amino acid sequence can be a naturally occurring nucleic acid sequence or a non-naturally occurring amino acid sequence.

[0031] In the next step of the process according to the invention, it is necessary to bring the biological sample into contact with the polypeptide and incubate it. Within the scope of the invention, it is of secondary importance whether the biological sample is added first and the polypeptide second, or vice versa. This "bringing into contact" can be achieved, for example, by dripping, injecting, applying, filling, inserting, and / or by any other method known to those skilled in the art for bringing the polypeptide into contact with the biological sample.

[0032] The term "incubation" in connection with the process according to the invention refers to a process step in which the biological sample is in direct contact with the polypeptide for a certain period of time. It is conceivable that the incubation takes place over a certain period and / or at a specific temperature. This is known to those skilled in the art. Furthermore, this process step can include not only incubating the biological sample with the polypeptide but also monitoring it. The method of monitoring is sufficiently known to those skilled in the art and can, for example, be based on a defined time period or on the experience of those skilled in the art, for example, by observing the biological sample. Alternatively, a colored or otherwise visually visible indication can be provided. This can be achieved, for example, by means of a chemical and / or biochemical indicator, e.g.,This can be achieved by means of staining, a color change of the stain, or loss of staining. The steps of incubation and monitoring can be performed alternately and repeated.

[0033] In the next step of the method according to the invention, it is necessary to determine an anti-blood group antibody, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 or more identical or different anti-blood group antibodies, as described elsewhere, and / or an anti-CD38 antibody, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 or more identical or different anti-CD38 antibodies, as described elsewhere, in the biological sample. It is conceivable that this determination includes determining the antibody strength. This step serves, among other things, to determine the blood group described in detail elsewhere, in particular the antigens in the Kell blood group system, or to differentiate between the known blood groups, in particular between the main antigens Kell and Cellano, as well as the anti-blood group antibodies and / or anti-CD38 antibodies contained in the biological sample.Simultaneously, this step detects the binding of the antibody and / or polypeptide to the erythrocyte membranes from the biological sample. It is understandable to a person skilled in the art that the determination is preferably carried out semi-quantitatively, quantitatively, directly, and / or indirectly using methods known from the prior art, such as an algorithm, and / or a detection method, in particular the analysis of the detection method, such as a gel, an assay, such as an enzyme-linked immunosorbent assay (ELISA), an agglutination reaction, in particular a staged agglutination reaction, and / or an indirect antiglobulin test (IAT), such as antibody detection tests (screening with test panels), antibody identification panels, and / or antihuman globulins (AHG). In this regard, it is conceivable to add an agglutination-promoting substance, such as an anti-human antibody, to the aforementioned detection methods.

[0034] Furthermore, it is conceivable that the agglutination determination is carried out by applying the reaction mixture of polypeptide and biological sample to a microcolumn, such as a gel card and / or cassette, and layering it for blood group determination, observing the movement of the erythrocytes through the bed of gel particles, preferably after increasing the force of gravity by centrifugation to move the erythrocytes through the bed of gel particles. The gel card is a polyacrylamide-based gel card known from the prior art, such as a DG gel, an ID card, a BioVue, or a column gel card, such as a 6- or 8-column gel card based on column agglutination technology. It is known to those skilled in the art that a gel card contains microtubes filled with a polyacrylamide gel matrix, which act as a sieve.Furthermore, it is conceivable to perform the determination using multicard tests, solid-phase erythrocyte adherence tests, gel-based tests and / or blood group analyzers.

[0035] It is understood by a person skilled in the art that a determination cannot usually be 100% accurate. The term therefore refers to a statistically significant probability regarding the accuracy of the determination of the anti-blood group antibody and thus the blood group, and / or regarding the accuracy of the determination of the anti-CD38 antibody. Whether such a determination is statistically significant can be determined by a person skilled in the art without inventive steps using methods known in the field. Examples include statistical evaluation tools such as determining the confidence interval, the p-value, the Student's st test, the Mann-Whitney U test, etc. The corresponding intervals are at least 90%, 95%, 97%, 98%, or 99% accurate. The p-values ​​are preferably 0, 1, 0.05, 0.01, 0.005, or 0.0001.Preferably, within the scope of the present invention, this determination is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or correct.

[0036] The analytical in vitro method according to the invention therefore serves to detect an antibody directed against a blood group antigen and / or an anti-CD38 antibody contained in the biological sample by bringing the biological sample to be analyzed, comprising erythrocytes, into contact with a polypeptide. The method may include additional steps that lie after or between the explicitly listed essential steps a) to d). Furthermore, it is conceivable that individual steps may be repeated as often as desired. Preferably, the method is automatable and can be performed in multicard tests, solid-phase erythrocyte adherence tests, gel-based tests, and / or blood group analyzers.

[0037] Within the scope of the present invention, it has been recognized that the biological sample treated with a therapeutic anti-CD38 antibody must be neutralized in order to enable the reliable determination of an anti-blood group antibody in the treated sample. The term "neutralize" means that the cross-reactions occurring in the aforementioned detection methods, such as indirect antiglobulin tests, such as consistently positive agglutination reactions, and / or interfering factors, such as interference signals in indirect anti-human globulin tests, are eliminated or suppressed in the biological sample treated with a therapeutic anti-CD38 antibody.Preferably, cross-reactions and / or interfering signals are eliminated in such a way that the biological sample treated with a therapeutic anti-CD38 antibody shows no significant, and in particular no, interference in indirect antiglobulin assays, especially no daratumumab- and isatuximab-mediated interferences such as a positive agglutination reaction, thus allowing interference-free determination of blood group antibodies. This is preferably achieved by the polypeptide (for example, in its entirety or a fragment thereof, as described elsewhere) occupying the corresponding CD38 binding sites on the erythrocytes in order to subsequently and / or simultaneously prevent the binding of therapeutic anti-CD38 antibodies such as daratumumab, isatuximab, mezagitamab, erzotabart, CM313, and / or felzartamab. In this way, neutralization is achieved.Without prior neutralization of therapeutic anti-CD38 antibodies, false-positive results are expected in tests such as blood group serological examinations, making their correct interpretation impossible, for example, in indirect antiglobulin tests. According to the invention, neutralization is achieved using the provided polypeptide. Thus, the method according to the invention makes it possible to determine an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample in a simple, cost-effective, and reliable manner, regardless of whether the biological sample has been treated with a therapeutic anti-CD38 antibody.In this way, it is possible to quickly and reliably determine a patient's blood type, as well as the antibodies against a specific blood type, with sufficient accuracy in just one test. This is useful, for example, to clarify potential incompatibilities and / or adverse effects before a blood transfusion and / or antibody therapy, as well as for conducting a blood compatibility test. This eliminates the additional costs associated with repeat tests or lengthy laboratory analyses. Furthermore, it has proven advantageous that the biological sample is not directly or indirectly affected during the determination of anti-blood group antibodies, as the erythrocytes are brought into contact with the polypeptide. This also means that the procedure is very gentle for the patient, as it spares them the need for repeated sample collection.

[0038] Advantageous further developments of the invention, which can be implemented individually or in combination, are presented in the dependent claims.

[0039] In a further development step, it is conceivable that a sample is provided containing an anti-CD38 antibody, wherein the anti-CD38 antibody is specific for CD38, and that in step c) the sample is brought into contact with and incubated with the biological sample before, during, and / or after the biological sample is brought into contact with and incubated with the polypeptide. It is understandable to a person skilled in the art that a pre-block is a process of bringing the sample into contact with and incubating the biological sample, where the sample is added shortly after the biological sample has been brought into contact with and incubated with the polypeptide, i.e., after a short incubation period. However, it is also conceivable that the sample is added to the polypeptide and that this mixture is then brought into contact with and incubated with the biological sample (so-called post-block).Furthermore, it is understandable to a specialist that a co-block is the process of bringing the sample into contact with and incubating it with the biological sample, which occurs simultaneously with, or during, the contact and incubation of the biological sample with the polypeptide. This means that in a co-block, the sample and the polypeptide are brought into contact and incubated simultaneously with the biological sample. In this way, it is also possible to provide a method for blocking, inhibiting, and / or neutralizing an anti-CD38 antibody in a biological sample.

[0040] It is conceivable that the specific anti-blood group antibody is an antibody against an antigen from a blood group or blood group system selected from ABO, MNS, P1PK, Rhesus (RhD; RhCE), Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Cartwright (Yt), Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H system, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, John Milton Hagen, I system, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, Sid, CTL2, PEL, MAM, EMM, ABCC1, Er, CD36, ATP11C and MAL, as well as those belonging to the blood group collections Cost, li, Er, 209, 210 and such as the low- and high-incidence antigens of the 700 series, such as Batty, Christiansen, Biles, Box, Torkildsen, Peters, Reid, Jensen, Livesay, Milne, Rasmussen and Jones, and the 901 series, such as ABTI and LKE.Furthermore, it is conceivable that the specific anti-CD38 antibody is selected from daratumumab, felzartamab, mezagitamab, erzotabart, CM313, and / or isatuximab. In this way, it is possible to determine a large number of potential antibodies in a single biological sample, thus completely eliminating additional costs for any repeat tests or lengthy laboratory analyses, for example, to clarify a possible incompatibility and / or impairment before a blood transfusion and / or antibody therapy, as well as to perform a blood compatibility test. Moreover, the patient is spared the need for repeated sample collection.

[0041] Within the scope of the invention, it is further conceivable that the concentration of the anti-CD38 antibody to be blocked, inhibited and / or neutralized, for example in the sample and / or in the biological sample, is between 0.001 pg / ml and 3500 pg / ml. Preferably, the concentration of the anti-CD38 antibody to be blocked, inhibited, and / or neutralized is at least 0.001 pg / ml, 0.002 pg / ml, 0.003 pg / ml, 0.004 pg / ml, 0.005 pg / ml, 0.006 pg / ml, 0.007 pg / ml, 0.008 pg / ml, 0.009 pg / ml, 0.01 pg / ml, 0.02 pg / ml, 0.03 pg / ml, 0.04 pg / ml, 0.05 pg / ml, 0.06 pg / ml, 0.07 pg / ml, 0.08 pg / ml, 0.09 pg / ml, 0.1 pg / ml, 0.2 pg / ml, 0.3 pg / ml, 0.4 pg / ml, 0.5 pg / ml, 0.6 pg / ml, 0.7 pg / ml, 0.8 pg / ml, 0.9 pg / ml, 1 pg / ml, 2 pg / ml, 3 pg / ml, 4 pg / ml, 5 pg / ml, 6 pg / ml, 7 pg / ml, 8 pg / ml, 9 pg / ml

[0042] 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml, 100 pg / ml, 200 pg / ml, 300 pg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, 800 pg / ml, 900 pg / ml, 1000 pg / ml, 1100 pg / ml, 1200 pg / ml, 1300 pg / ml, 1400 pg / ml, 1500 pg / ml

[0043] 1600 pg / ml, 1700 pg / ml, 1800 pg / ml, 1900 pg / ml, 2000 pg / ml,

[0044] 2100 pg / ml, 2200 pg / ml, 2300 pg / ml, 2400 pg / ml, 2500 pg / ml,

[0045] 2600 pg / ml, 2700 pg / ml, 2800 pg / ml, 2900 pg / ml, 3000 pg / ml,

[0046] 3100 pg / ml, 3200 pg / ml, 3300 pg / ml, 3400 pg / ml or 3500 pg / ml.

[0047] In a further development, it is conceivable that the biological sample is human or animal blood or parts thereof, such as blood plasma and / or blood serum. This is characterized by good availability combined with high storability and transportability. Furthermore, it is conceivable that in step b) at least 2, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more, identical or different polypeptides are provided, which are specific for CD38 and have an amino acid sequence described elsewhere. It is conceivable that these polypeptides represent different clones of a polypeptide, which bind monovalently to certain epitopes of the CD38 antigen.Preferably, each of the at least two polypeptides forms at least one paratope with high specificity for the CD38 antigen, such that the at least two polypeptides preferably form at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more, identical or different paratopes with high specificity for the CD38 antigen. It is conceivable that the at least two polypeptides possess two binding sites for the CD38 antigen, thus forming two different paratopes. This offers the advantage that these two polypeptides can specifically block, inhibit, and / or neutralize the binding of two or more of the therapeutic anti-CD38 antibodies mentioned elsewhere, such as daratumumab and isatuximab.Preferably, the at least two polypeptides are provided as a mix, so that within the scope of the invention it is understandable that the mix of at least two polypeptides is referred to as the “blocker mix”.

[0048] Furthermore, it is conceivable that the polypeptide in step b) and / or in step c), in particular the recombinantly soluble form of a single-do-main polypeptide and / or a previously described mix, is present in a concentration of 0.01 pg / ml to 3000 pg / ml. Preferably, the polypeptide is present in a concentration of at least 0.01 pg / ml, 0.02 pg / ml, 0.03 pg / ml, 0.04 pg / ml, 0.05 pg / ml, 0.06 pg / ml, 0.07 pg / ml, 0.08 pg / ml, 0.09 pg / ml, 0.1 pg / ml, 0.2 pg / ml, 0.3 pg / ml, 0.4 pg / ml, 0.5 pg / ml, 0.6 pg / ml, 0.7 pg / ml, 0.8 pg / ml, 0.9 pg / ml, 1 pg / ml, 2 pg / ml, 3 pg / ml, 4 pg / ml, 5 pg / ml, 6 pg / ml, 7 pg / ml, 8 pg / ml, 9 pg / ml.

[0049] 10 pg / ml, 15 pg / ml, 20 pg / ml, 25 pg / ml, 30 pg / ml, 35 pg / ml, 40 pg / ml, 45 pg / ml, 50 pg / ml, 55 pg / ml, 60 pg / ml, 65 pg / ml, 70 pg / ml, 75 pg / ml, 80 pg / ml, 85 pg / ml, 90 pg / ml, 95 pg / ml, 100 pg / ml, 150 pg / ml

[0050] 200 pg / ml, 250 pg / ml, 300 pg / ml, 350 pg / ml, 400 pg / ml, 450 pg / ml, 500 pg / ml, 550 pg / ml, 600 pg / ml, 650 pg / ml, 700 pg / ml, 750 pg / ml, 800 pg / ml, 850 pg / ml, 900 pg / ml, 950 pg / ml, 1000 pg / ml, 1100 pg / ml, 1200 pg / ml, 1300 pg / ml, 1400 pg / ml, 1500 pg / ml, 1600 pg / ml

[0051] 1700 pg / ml, 1800 pg / ml, 1900 pg / ml, 2000 pg / ml, 2100 pg / ml,

[0052] 2200 pg / ml, 2300 pg / ml, 2400 pg / ml, 2500 pg / ml, 2600 pg / ml,

[0053] 2700 pg / ml, 2800 pg / ml, 2900 pg / ml or 3000 pg / ml.

[0054] Furthermore, it is conceivable that the incubation in step c), i.e., of the biological sample, the polypeptide and / or the sample, takes place at a temperature of 0°C to 42°C for a duration of 0 minutes to 60 minutes. Incubation is preferably carried out at a temperature of at least 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C 40°C, 41°C or 42°C.

[0055] Preferably, incubation is carried out for a duration of at least 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes. It is understood by a person skilled in the art that incubation for 0 minutes is referred to as a co-block. Furthermore, it is understood by a person skilled in the art that if the sample is first brought into contact with the biological sample and incubated for a short time, i.e., for at least 0 minutes or more, and then the polypeptide is added, the procedure is referred to as a post-block. In this way, an improvement in the determination of the anti-blood group antibody and / or the anti-CD38 antibody in the biological sample can be achieved.

[0056] Furthermore, it is conceivable that the incubation in step c) includes pre-incubation of the biological sample, the polypeptide and / or the sample, with the pre-incubation taking place at a temperature of 0°C to 42°C for a duration of 0 minutes to 120 minutes. Pre-incubation is preferably carried out at a temperature of at least 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C 40°C, 41°C, or 42°C. Pre-incubation is preferably carried out for a duration of at least 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes. It is understood by those skilled in the art that an incubation period of 0 minutes is referred to as a co-block.This method improves the determination of anti-blood group antibodies and / or anti-CD38 antibodies in the biological sample, as the biological sample, the polypeptide, and / or the sample have a longer overall incubation time. Furthermore, pre-incubation causes the therapeutic anti-CD38 antibodies to be masked on the erythrocytes, particularly the erythrocyte membrane, as described elsewhere. This partially or completely blocks, inhibits, and / or neutralizes the binding of the therapeutic anti-CD38 antibodies from the biological sample to the CD38 antigen, as described elsewhere.

[0057] It is assumed that the definition and / or explanations of the above-mentioned terms apply to all aspects described below, unless otherwise stated. The invention further proposes a device for carrying out the method described elsewhere and thus for determining an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample. The device has a receptacle for a container comprising a biological sample and a polypeptide. Preferably, the container also includes a sample comprising an anti-CD38 antibody.Furthermore, the device includes an analysis, evaluation and / or assessment unit for determining an anti-blood group antibody and / or the anti-CD38 antibody, wherein the biological sample comprises erythrocytes, wherein the erythrocytes express a transport protein (CD38), and wherein the polypeptide, as well as the anti-CD38 antibody, is specific for CD38.

[0058] The term "device" is familiar to those skilled in the art and refers to a system comprising the aforementioned units, which are operationally linked to enable the determination of the anti-blood group antibody and / or the anti-CD38 antibody in the biological sample according to the method of the invention. It is conceivable that the device may include further units, such as a temperature control device, like a cooling and / or heating device, a timekeeping device, like a clock, and / or a contacting device, pre-incubation device, and / or incubation device for the biological sample, the sample, and / or the polypeptide, such as a pipette, a tube, and / or other structural (separation) device. Preferably, the device is automatable. More preferably, the device is a semi- or fully automatic blood group analyzer.

[0059] The term "analysis unit" refers to any means suitable for carrying out the method according to the invention and thus for determining the anti-blood group antibody and / or the anti-CD38 antibody in the biological sample. Preferably, the determination is carried out according to the described method. Suitable analysis units are known to a person skilled in the art, such as a computer and / or a computer program, a detector and / or a detection unit.

[0060] The term "evaluation and / or assessment unit" refers to a data processing unit suitable for determining and / or evaluating the determination of the anti-blood group antibody and / or the anti-CD38 antibody in the biological sample.

[0061] Suitable evaluation and / or analysis units are known to a person skilled in the art, such as a computer and / or a computer program with an implemented algorithm for performing the evaluation. A computer program can also assess the result of the evaluation. It is understood that the result may require interpretation by a specialist. However, expert system devices are also provided where the output comprises processed raw data, preferably as absolute or relative quantities, the interpretation of which does not require a specialist.

[0062] According to the invention, a kit for carrying out the method described elsewhere, and thus a kit for determining an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample, is further proposed, comprising: a) a container comprising a biological sample which, as described elsewhere, comprises erythrocytes, wherein the erythrocytes express a transport protein (CD38), and a polypeptide which is specific for CD38; and

[0063] b] Instructions for carrying out the procedure.

[0064] The term "kit" is familiar to those skilled in the art and refers to a collection of the aforementioned components (kit-of-parts), which are preferably provided separately or within a single container. It is conceivable that the container additionally includes a sample comprising an anti-CD38 antibody, wherein the anti-CD38 antibody is specific for CD38. Furthermore, it is conceivable that the kit also includes a detection agent known from the prior art for detecting the anti-blood group antibody and / or the anti-CD38 antibody in the biological sample.

[0065] The term "instructions for carrying out the method" refers to a numerical, written, and / or graphical representation of the description by means of which a simplification of the execution of the method according to the invention is achieved. These may preferably be in the form of a manual or may be provided by a computer program, such as an application. Furthermore, it is conceivable that the computer program includes an implemented algorithm that is capable of carrying out, evaluating, and / or assessing the determination referred to in the methods of the present invention. The computer program may be provided on a data storage medium or device such as an optical storage medium (e.g., a compact disc) or directly on a computer or data processing device. In addition, the instructions may preferably include standards for quantities of use as known to a person skilled in the art.Furthermore, the instructions include requirements for the storage and disposal of the container and the biological sample.

[0066] According to the invention, an expression system, in particular a recombinant expression system, for the production of a polypeptide is further proposed. The polypeptide has an amino acid sequence described elsewhere and is specific for a transport protein (CD38). The amino acid sequence is selected from the group comprising: i) an amino acid sequence according to SEQ ID No. 1 to SEQ ID No. 58, which codes for an amino acid molecule; and

[0067] ii) an amino acid sequence encoding an amino acid molecule that is at least 50% identical to the amino acid molecule encoded by the amino acid sequence from i); and

[0068] iii) an amino acid sequence for a fragment of an amino acid molecule from i) or ii), wherein the fragment encodes a polypeptide, and

[0069] where the expression system is either a eukaryotic expression system or a prokaryotic expression system.

[0070] The term "expression system" is familiar to those skilled in the art and refers to a biological system capable of targeted and controlled protein biosynthesis, that is, producing, or expressing, specific proteins or polypeptides based on a nucleic acid template. This can refer to a transient and / or stable expression system. According to the invention, the expression system, in particular the recombinant expression system, is a eukaryotic expression system, such as Saccharomyces cerevisiae, Baby Hamster Kidney fibroblasts (BHK), NSO, Sp2 / 0, Chinese Hamster Ovary (CHO), Chinese Hamster Ovary-Kl (CHO-Kl), Human Embryonic Kidney (HEK), HT-10780, PER. C6, Henrietta Lacks (HeLa) or Jurkat cells, or a prokaryotic expression system such as BL21 (DE3) from E. coli, so that the expression system can be a classical plasmid or a vector in the sense of a retrovirus, which integrates into the genome of the host cell.Preferably, the expression system is an expression vector. Using the expression system, it is possible to synthesize a polypeptide described elsewhere. Preferably, it is possible to synthesize more than one polypeptide. In this way, it is possible to obtain a stable cell line.

[0071] Within the scope of the invention, it has been recognized that, for the production of the polypeptide, as described elsewhere, corresponding nucleotide sequences were used which are suitable for the production of a polypeptide comprising an amino acid sequence according to SEQ ID No. 1 to SEQ ID No. 58. The development, provision, and use of such nucleotide sequences are within the scope of skilled craftsmanship. It is conceivable, for example, that the amino acid sequence of the corresponding polypeptide according to SEQ ID No. 1 to SEQ ID No. 58 is converted into a codon-optimized nucleic acid sequence using appropriate software tools. The synthesized gene is then cloned in a eukaryotic expression system or a prokaryotic expression system, such as a CHO-Kl-compatible expression vector. Furthermore, it is conceivable that the genes designed in silico were subsequently produced via gene synthesis and cloned into an expression vector.

[0072] In a further development, it is conceivable that the polypeptide has an N- and / or C-terminal affinity tag, such as a GST, SUMO, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, histidine tag (His-tag), Myc-tag, NE-tag, S-tag, SBP-tag, Softag 1, Softag 3, Strep-tag, TC tag, V5 tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, BCCP (Biotin Carboxyl Carrier Protein), glutathione S-transferase-tag, green fluorescent protein-tag, other fluorescent protein tags, HaloTag, maltose binding protein-tag, Nus-tag, or thioredoxin-tag. Preferably, the polypeptide has 2, 3, 4, 5, 6, 7, 8, 9, 10, or more identical or different affinity tags.

[0073] Preferably, the polypeptide is linked to the affinity tag via a linker, such as a GS linker. This offers the advantage of simplified purification of the polypeptide produced by the expression system, for example, by affinity chromatography using FLAG-tag, Streptactin, IMAC, or chromatography columns. Preferably, the affinity tag has a cleavage sequence to facilitate its removal from the polypeptide.

[0074] It is conceivable that the cleavage sequence is a TEV cleavage sequence recognizable by Tobacco Etch Virus (TEV) protease, a thrombin cleavage sequence recognizable by thrombin endoprotease, and / or an HRV 3C cleavage sequence recognizable by Human Rhenovirus 3C (HRV 3C). The cleavage sequence can be located at the N- and / or C-terminus. Furthermore, affinity day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more preferably exhibits identical or different cleavage sequences.

[0075] In a further development, it is conceivable that the polypeptide is a mono-specific, bi-specific, tri-specific, or multi-specific single-domain polypeptide and / or that the polypeptide is monoclonal, polyclonal, or a combination thereof. The advantages of this design are known to those skilled in the art and are described elsewhere.

[0076] According to the invention, a polypeptide is further proposed which is produced using the expression system described elsewhere. The polypeptide blocks, inhibits and / or neutralizes at least one binding site, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or all binding sites, and / or the binding of an anti-CD38 antibody, in particular to erythrocytes, wherein the erythrocytes express a transport protein (CD38), partially or completely, as described elsewhere.

[0077] In a further development, it is conceivable that the anti-CD38 antibody is selected from daratumumab, felzartamab, mezagitamab, erzotabart, CM313 and / or isatuximab, as described elsewhere. According to the invention, a polypeptide, as described elsewhere, and / or a pharmaceutical composition comprising a polypeptide described elsewhere and at least one further component for use in the determination of an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample comprising erythrocytes, wherein the erythrocytes express a transport protein (CD38), is further proposed, as described elsewhere.

[0078] The term "pharmaceutical composition" as used here refers to a mixture of at least one polypeptide, as described in detail elsewhere, and at least one other component. Preferably, such other components may be stabilizers, wetting agents, colorants, preservatives such as Pro-Clean 300 and / or sodium azide, pharmaceutical carriers, pharmaceutically acceptable carriers, diluents, pharmaceutically acceptable diluents, additional pharmaceutical agents, anti-caking agents, and the like. Preferred diluents include water, alcohols, physiological saline solutions, buffers such as a biocompatible buffer or phosphate-buffered saline, histidine-buffered saline, citrate-buffered saline, HEPES-buffered saline, an extremophilic preservative such as ectoine, syrups such as trehalose, sucrose, or maltose, oil, water, emulsions, various types of wetting agents, and the like.The carrier must be acceptable in the sense that it is compatible with the other components of the composition and that it is not harmful to the patient's organism. The pharmaceutical carrier used may be a solid, a gel, or a liquid. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and similar substances. Similarly, the carrier or diluent may contain a time-retardant material well-known in the field, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. These suitable carriers include those mentioned above and others known in the field, such as those published by Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, the European Pharmacopoeia, the Homeopathic Pharmacopoeia of the USA, or HAB.The pharmaceutically acceptable diluent is chosen so as not to impair the biological activity of the combination. Examples of such diluents include distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. Furthermore, the pharmaceutical composition may also contain other carriers, excipients, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. The polypeptide and / or pharmaceutical composition must be adapted for the intended use.

[0079] According to the invention, a polypeptide, as described elsewhere, and / or a pharmaceutical composition comprising a polypeptide described elsewhere and at least one further component for use in blocking, inhibiting, and / or neutralizing an anti-CD38 antibody in a biological sample comprising erythrocytes, wherein the erythrocytes express a transport protein (CD38), is proposed, as described elsewhere. This is important, for example, but by no means exclusively, for clarifying a possible incompatibility and / or impairment prior to a blood transfusion and / or antibody therapy of a patient, as well as for performing a blood compatibility test in a patient.

[0080] According to the invention, a polypeptide, as described elsewhere, and / or a pharmaceutical composition comprising a polypeptide described elsewhere and at least one further component for use in a therapeutic or non-therapeutic procedure for blocking, inhibiting, and / or neutralizing an anti-CD38 antibody in a biological sample comprising erythrocytes, wherein the erythrocytes express a transport protein (CD38), is proposed as described elsewhere. Such therapeutic or non-therapeutic procedures include, for example, but by no means exclusively, those for investigating a possible incompatibility and / or impairment prior to a blood transfusion and / or antibody therapy, as well as for performing a blood compatibility test (crossmatch).

[0081] Further details, features, and advantages of the invention will become apparent from the following description of the preferred embodiments in conjunction with the dependent claims. The respective features can be implemented individually or in combination with one another. The invention is not limited to the embodiments shown. The embodiments are illustrated schematically in the figures.

[0082] Specifically, we show:

[0083] Fig. 1 - 22 is a tabular overview of the results of the embodiments 1 - 22 described elsewhere.

[0084] The following exemplary embodiments serve only to illustrate the invention. They are not intended to limit the subject matter of the patent claims in any way.

[0085] Examples:

[0086] Example 1: Single-domain blocker variants inhibit anti-CD38 antibodies in indirect antiglobulin tests.

[0087] Sample preparation: 50 pl of a 0.8% cell suspension #2 (Data-Cyte, specifically Data-Cyte Plus 3%, Grifols; Lot#: 610024017) with 25 pl of single domain blocker blocker (Blocker #1 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18) or #2 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 4)) were pre-incubated on red blood cells (RBCs) for 2 min at room temperature. Subsequently, 25 pl of daratumumab- or isatuximab-containing ABneg sera from a patient (monoclonal antibody 800 pg / ml) were added and incubated for 30 min at 37°C.

[0088] Subsequently, antibody screening was performed on a gel card (ID card, Lot#: 9065014201), which had previously been centrifuged in an ID card centrifuge. The agglutination results can be read in Fig. 1, where a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicates complete inhibition of the binding of the present anti-CD38 antibody. Pre-incubation of the single-domain blockers with the RBCs completely eliminated interference in the IAT for daratumumab and isatuximab (see Fig. 1). In this way, it was shown, as can also be seen in Fig. 1, that the tested single domain blockers #1 and #2 neutralize the anti-CD38 antibodies daratumumab and isatuximab in artificial sera and thus completely eliminate the effect of therapeutic anti-CD38 antibodies in the agglutination test.

[0089] Example 2: Titration of single-domain blocker variants to determine the degree of neutralization of anti-CD38 antibodies

[0090] Sample preparation: 50 pl of a 0.8% cell suspension #2 (Data-Cyte, specifically Data-Cyte Plus 3%, Grifols; Lot#: ) were prepared.

[0091] 9065014201) with 25 pl single domain blocker-blocker (Blocker #1 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18) or #2 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 4)) of various dilutions was pre-incubated for 2 min at room temperature on RBCs. Subsequently, 25 pl daratumumab- or isatuximab-containing ABneg sera from a patient (monoclonal antibody 800 pg / ml) were added and incubated for 30 min at 37°C.

[0092] Subsequently, antibody screening was performed on a gel card (ID card, Lot#: 9065014201), which had previously been centrifuged in an ID card centrifuge. The agglutination results can be read in Fig. 2, where a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicates complete neutralization of the binding of the present anti-CD38 antibody. Pre-incubation of the single-domain blockers with the RBCs completely eliminated the interference in the IAT for daratumumab and isatuximab at a dilution of 1 / 8 and Yi, respectively (see Fig. 2A and 2B). In this way, it was shown, as can also be seen in Fig. 2, that the tested single domain blockers #1 and #2 neutralize the anti-CD38 antibodies daratumumab and isatuximab in artificial sera in a concentration-dependent manner, thus completely eliminating the effect of therapeutic anti-CD38 antibodies in the agglutination test.

[0093] Example 3: Titration of the Single Domain Blocker Mix 12 to determine the degree of neutralization of anti-CD38 on gel cards

[0094] Sample preparation: 50 pl of a 0.8% cell suspension #2 (Data-Cyte, in particular Data-Cyte Plus 3%, Grifols; Lot#: 9065014201 for Fig. 3A) or a 0.8% cell suspension #3 (Identisera Diana 0.8%; Lot#: 24019 for Fig. 3B) with 25 pl or 10 pl of Single Domain Blocker-Blockermix 12 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18 and a polypeptide with an amino acid sequence according to SEQ ID No. 4) of various dilutions (up to 1:2187 for Fig. 3A, up to 1:64 for Fig. 3B) were pre-incubated on RBCs for 2 min at room temperature. Subsequently, 25 pl of ABneg sera containing daratumumab or isatuximab (monoclonal antibody 3200 pg / ml) from a patient were added and incubated for 15 min at 37°C. Antibody screening was then performed on a gel card (ID card, Lot#: 9065014201 in Fig. 3A and DG gel card, Lot#: 3009280 in Fig. 3B) that had previously been centrifuged.The agglutination results can be read in Figures 3A and 3B, where a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicates complete neutralization of the binding of the existing anti-CD38 antibody. Pre-incubation of the single-domain blocker-blocker mix 12 with the RBCs completely eliminated the interference in the IAT for daratumumab and isatuximab at dilutions of 1:81 (see columns A7 and B7 in Figure 3A) and 1:8 and 1:2, respectively (see columns A5 and B3 in Figure 3B). In this way, it was shown that the tested single-domain blocker-blocker mix 12 neutralizes the anti-CD38 antibodies daratumumab and isatuximab in artificial sera in a concentration-dependent manner, thus completely eliminating the effect of therapeutic anti-CD38 antibodies in the agglutination test. This neutralization works on various gel cards.

[0095] Example 4: Detection of irregular antibodies in the presence of daratumumab or isatuximab

[0096] Sample preparation: 50 pl of each 0.8% cell suspension #1 - #11 (Identisera Diana 0.8%, Grifols; Lot#: 24019) were pre-incubated with 10 pl of Single Domain Blocker-Blockermix 12 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18 and a polypeptide with an amino acid sequence according to SEQ ID No. 4) at a dilution of 1:2 for 2 min at room temperature on RBCs. Subsequently, 25 pl of anti-Kell-positive sera containing daratumumab or isatuximab (monoclonal antibody (MAB) 800 pg / ml; Fig. 4A) or 25 pl of sera containing daratumumab or isatuximab with irregular blood group antibodies of various specificities (MAB: 800 pg / ml; Fig. 4B) were added and incubated for 15 min at 37°C. A negative and positive control (ABneg serum) served as the respective controls (column 1 in Fig. 4A and 4B: without MAB, and column 2 in Fig. 4B: without MAB).

[0097] 4A and 4B: with MAB addition). Anti-Kell-mediated agglutination was performed in column 14 in Fig. 4A (without MAB addition). Anti-Rhesus D-mediated agglutination was performed in columns 3A / 3B and 4A / 4B in Fig. 4B (with MAB addition). Anti-Fya-mediated agglutination was performed in columns 5A / 5B and 6A / 6B in Fig. 4B (with MAB addition). Anti-Rhesus c-mediated agglutination was performed in columns 7A / 7B and 8A / 8B in Fig. 4B (with MAB addition).

[0098] Subsequently, antibody screening was performed on a gel card (DG-Gel-Card, Lot#: 3009280), which had previously been centrifuged in a DG-Gel-Card centrifuge. The agglutination results can be read in Figures 4A and 4B, with a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicating complete neutralization of the binding of the present anti-CD38 antibody. Pre-incubation of the single-domain blocker-blocker mix 12 with the RBCs completely eliminated daratumumab- and isatuximab-mediated interference (see Figures 4A and 4B). Simultaneously, the respective irregular antibodies were correctly detected in the antibody screening test (see columns 3 and 9 in Figure 4A and columns 4, 6, and 8 in Figure 4B).

[0099] In this way, it was shown that the tested single domain blocker mix 12 neutralizes the interference caused by the anti-CD38 antibody daratumumab or isatuximab in various irregular sera on DG gel cards and that the irregular blood group antibody can be correctly detected on the corresponding test cells (- / +) in the agglutination test.

[0100] Example 5: Neutralization potential of single domain blocker #1 in the pre- and co-block approach. Sample preparation: 50 gl of a 0.8% cell suspension #2 (Data-Cyte, especially Data-Cyte Plus 3%, Grifols; Lot#: ) were used.

[0101] 9065014201) with 25 pl of Single Domain Blocker-Blocker #1 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18) of various dilutions up to 1:27 at room temperature on RBCs pre-incubated (pre-block) or co-incubated in parallel with the MAB-containing serum (co-block). For the samples of the pre-block reaction, 25 pl of daratumumab-containing ABneg sera (monoclonal antibody (MAB) 3200 pg / ml) were added 2 min after the start of the pre-block, which were incubated for 15 min at 37°C. For the samples of the co-block approach, 25 pl of Daratumumab / Single Domain Blocker-Blockers #l-containing ABneg sera (monoclonal antibody (MAB) 3200 pg / ml) were added directly and incubated for 15 min at 37°C.

[0102] Subsequently, antibody screening was performed on a gel card (ID card, Lot#: 9065014201) that had been previously centrifuged in an ID card centrifuge. The agglutination results can be read in Fig. 5, where a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicates complete neutralization of the binding of the present anti-CD38 antibody. While the pre-block approach eliminated daratumumab-mediated interference for all tested dilutions, the co-block approach, in contrast, was only able to eliminate interference at no dilution (see Fig. 5). Thus, the neutralization potential of the single-domain blocker blocker #1 in the presence of the anti-CD38 MAB daratumumab was demonstrated in a pre- or co-block approach on ID cards.

[0103] Example 6: Comparison of incubation temperatures for the inhibition of anti-CD38

[0104] Sample preparation: 50 pl of a 0.8% cell suspension #2 (Data-Cyte, specifically Data-Cyte Plus 3%, Grifols; Lot#:9065014201) was mixed with 25 pl of Single Domain Blocker-Blockermix 12 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18 and a polypeptide with an amino acid sequence according to SEQ ID No.

[0105] 4) Different dilutions up to 1:27 were pre-incubated on RBCs for 2 min at appropriate temperature. Subsequently, 25 pl of ABneg sera containing daratumumab or isatuximab (monoclonal antibody (MAB) 3200 pg / ml) were added and incubated for 15 min at 4°C, 22°C or 37°C.

[0106] Subsequently, antibody screening was performed on a gel card (ID card, Lot#: 9065014201), which had previously been centrifuged in an ID card centrifuge. The agglutination results can be read in Fig. 6, where a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicates complete neutralization of the binding of the present anti-CD38 antibody. As can be seen in Fig. 6, no restriction of neutralization for daratumumab or isatuximab was observed after pre-incubation of the single domain blocker-blocker mix 12 with the RBCs at various incubation temperatures, up to the highest dilution of 1:27 (see columns 1-6 in 6A-C and columns 7-12 in 12A-C in Fig. 6).In this way, it was shown that the tested single domain blocker-blocker mix 12 neutralizes the anti-CD38 antibodies daratumumab or isatuximab at different incubation temperatures on ID cards and completely eliminates the effect of therapeutic anti-CD38 antibodies in the agglutination test.

[0107] Example 7: Neutralization of anti-CD38-MABs in patient serum (pre-block)

[0108] Sample preparation: 50 pl of a 0.8% cell suspension #5 (Data-Cyte, specifically Data-Cyte Plus 3%, Grifols; Lot#: 9065014201 for Fig. 7A or Identisera Diana 0.8%, Grifols; Lot#: 24019 for Fig. 7B) were mixed with 25 pl of Single Domain Blocker-Blocker Mix 32 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49 and a polypeptide with an amino acid sequence according to SEQ ID No. 4) at a dilution of 1:2 (Fig. 7A) or with 10 pl of Single Domain Blocker-Blocker Mix 12 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18 and a polypeptide with an amino acid sequence according to SEQ ID No. 4) at a dilution of 1:2 (Fig. 7B) for 2 The samples were pre-incubated on RBCs for 15 minutes at room temperature. Subsequently, 25 pl of selected patient sera (#1, #2, #4, #5, #6, #7) of different treatment doses were added and incubated for 15 minutes at 37°C.The patient sera were obtained from patients who had received anti-CD38 antibody therapy (e.g., in treatment for multiple myeloma, systemic light chain amyloidosis, or monoclonal gammopathies).

[0109] Subsequently, antibody screening was performed on a gel card (ID card, lot #: 9065014201 in Fig. 7A or DG gel cards, lot #: 3009280 in Fig. 7B), which had previously been centrifuged. The agglutination results can be read in Figs. 7A and 7B, with a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicating complete neutralization of the binding of the present anti-CD38 antibody.

[0110] Pre-incubation of the single domain blocker-blocker mix 32 (Fig. 7A) and the single domain blocker-blocker mix 12 (Fig. 7B) with the RBCs did not result in any impairment of neutralization for anti-CD38-MABs in the patient sera. As can be seen in Figs. 7A and 7B, this demonstrated that the tested single domain blocker-blocker mix 32 and the single domain blocker-blocker mix 12, when used alone, completely eliminated the effect of the therapeutic anti-CD38 antibodies in the agglutination test during the pre-block phase and neutralized them on the gel cards, even in patient sera from patients who had received anti-CD38 antibody therapy. Example 8: Neutralization of anti-CD38-MABs in patient serum (co-block)

[0111] Sample preparation: 50 gl of a 0.8% cell suspension #5 (Data-Cyte, specifically Data-Cyte Plus 3%, Grifols; Lot#: 24019) together with 10 pl of Single Domain Blocker-Blocker Mix 12 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18 and a polypeptide with an amino acid sequence according to SEQ ID No. 4) at a dilution of 1:2 and 25 pl of selected patient sera (#1, #2, #4, #5, #6, #7) were incubated for 15 min at 37°C by direct addition to the RBCs. The patient sera were obtained from patients who had received anti-CD38 antibody therapy (e.g., in treatment for multiple myeloma, systemic light chain amyloidosis, or monoclonal gammopathies).

[0112] Subsequently, antibody screening was performed on a gel card (DG-Gel-Cards, Lot#: 3009280), which had previously been centrifuged. The agglutination results can be read in Fig. 8, where a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicates complete neutralization of the binding of the present anti-CD38 antibody. It is clearly visible in Fig. 8 that no impairment of neutralization for anti-CD38-MABs in the patient sera was observed when using a co-blocker approach. Thus, it was demonstrated that the tested single-domain blocker-blocker mixture 12 completely eliminates the effect of the therapeutic anti-CD38 antibodies in the agglutination test using the co-blocker and neutralizes them on the gel card, even in patient sera from patients who had received anti-CD38 antibody therapy.

[0113] Example 9: Neutralization potential of Single Domain Blocker-Blocker Mix 12 with patient serum (in DG gel cards). Sample preparation: 50 gl of a 0.8% cell suspension #3 (Identisera Diana 0.8%, Grifols; Lot#: 24019) were pre-incubated with 10 pl of Single Domain Blocker-Blocker Mix 12 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18 and a polypeptide with an amino acid sequence according to SEQ ID No. 4) of various dilutions up to 1:64 for 2 min at room temperature on RBCs. Subsequently, 25 pl of diluted patient serum (#5; diluted 1:3 in PBS) was added and incubated for 15 min at 37°C. The patient sera were also obtained from patients who had received anti-CD38 antibody therapy (e.g., in treatment for multiple myeloma, systemic light chain amyloidosis, or monoclonal gammopathies).

[0114] Subsequently, antibody screening was performed on a gel card (DG-Gel-Cards, Grifols, Lot#: 3009280), which had previously been centrifuged. The agglutination results can be read in Fig. 9, where a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicates complete neutralization of the binding of the present anti-CD38 antibody. It is clearly visible in Fig. 9 that no restriction of neutralization for anti-CD38-MAB was observed after pre-incubation of the single domain blocker-blocker mix 12 with the RBCs, down to a dilution of 1:32.In this way, it was shown that the tested single domain blocker-blocker mix 12 completely eliminates the effect of the therapeutic anti-CD38 antibodies in the pre-block agglutination test and neutralizes them on the gel card, even in patient sera from patients who have received anti-CD38 antibody therapy.

[0115] Example 10: Neutralization potential of the single domain blocker-blocker mix 12 with patient serum (in Ortho BioVue cards)

[0116] Sample preparation: Red blood cells (RBCs) from a Jkb+ donor were diluted in saline solution according to the manufacturer's protocol. 40 µl of patient serum #5 were pipetted into the reaction cavity of the column (Ortho BioVue, Lot# IGC185H) with 10 µl of Single Domain Blocker-Blocker Mix 12 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18 and a polypeptide with an amino acid sequence according to SEQ ID No. 4) and with 10 µl of this 1% cell suspension. This mix was then incubated for 15 min at 37°C. Patient serum #5 was obtained from a patient who had received anti-CD38 antibody therapy (e.g., for treatment of multiple myeloma, systemic light chain amyloidosis, or monoclonal gammopathies).

[0117] Subsequently, an antibody screening was performed. The Ortho BioVue cards were centrifuged in a suitable centrifuge. The agglutination results can be read in Fig. 10, where a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicates complete neutralization of the binding of the present anti-CD38 antibody. It is clearly visible in Fig. 10 that incubation of the single domain blocker-blocker mix 12 with the RBCs and the patient serum did not impair the neutralization of anti-CD38 antibodies. Thus, it was demonstrated that the tested single domain blocker-blocker mix 12 completely eliminates the effect of therapeutic anti-CD38 antibodies in the agglutination test, even in patient serum from patients who had received anti-CD38 antibody therapy, and also neutralizes them on Ortho BioVue cards.

[0118] Example 11: Stability study of a single domain blocker mix 12

[0119] Sample preparation: 50 pl of a 0.8% cell suspension #2 (Data-Cyte, specifically Data-Cyte Plus 3%, Grifols; Lot#: ) were prepared.

[0120] 9065014201) with 25 pl Single Domain Blocker-Blockermix 12 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18 and a polypeptide with an amino acid sequence according to SEQ ID No.

[0121] 4) Different storage conditions (A: 2-8°C for 5 months; B: -80°C (reference; 5 months)) and dilutions up to 1:2187 were pre-incubated on RBCs for 2 min at room temperature. Subsequently, 25 pl of daratumumab-containing ABneg sera (monoclonal antibody (MAB) 3200 pg / ml) were added and incubated for 15 min at 37°C.

[0122] Subsequently, antibody screening was performed on a gel card (ID card, lot #: 9065014201), which had previously been centrifuged. The agglutination results can be read in Fig. 11, where a completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicates complete neutralization of the binding of the existing anti-CD38 antibody, particularly to the RBCs. It is clearly visible in Fig. 11 that pre-incubation of the single-domain blocker-blocker mix 12 with the RBCs completely eliminated the interference in the IAT for daratumumab at a dilution of 1:81 (see columns A7 and B7 in Fig. 11), regardless of storage conditions.In this way, it was shown that the tested Single Domain Blocker-Blockermix 12 exhibits its full effect regardless of storage conditions (A: 2-8°C for 5 months; B: -80°C (reference; 5 months)) and dilution levels.

[0123] Example 12: Blockers neutralize anti-CD38 antibody-mediated interference in IAT using pre-block, co-block, and post-block techniques.

[0124] Sample preparation: For the experiments, a 0.8% cell suspension was prepared from the respective panel cell #10, #1, or #5 (#10, Panocell 10, IMMUCOR INC, Norcross, USA, Lot: 16058; #1, Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025013; #5, Panocell 10, IMMUCOR INC, Norcross, USA, Lot: 24120) with a dilution medium (ID-Diluent 2, Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 964053 00 41). For the pre-block procedure with daratumumab, 50 pl of the 0.8% panel cell #10 (Panocell 10, IMMUCOR, INC., Norcross, USA, Lot: 16058) were used. USA, Lot: 16058) with 4 g of blocker (Blocker #1, containing a polypeptide with an amino acid sequence according to SEQ ID No. 18) for 2 min at room temperature in the upper cavity of the ID-Card LISS / Coombs anti-IgG + C3d (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot:

[0125] 993981 18 21) was incubated at room temperature. Subsequently, 25 pl of daratumumab-containing AB serum (daratumumab: 1000 pg / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: NIS5P00; Serum matrix: Human serum AB male, HIV tested, from BIOWEST SAS, Nuaille, France, Lot:

[0126] S00SS10015), which was incubated together with the RBC / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 18) for 15 min at 37°C. AB serum without anti-CD38 antibodies served as a control for the assessment of a negative agglutination reaction (column #1).

[0127] For the co-block procedure with daratumumab, 25 pl of the daratumumab-containing AB serum (daratumumab: 750 pg / ml; Stock: ) were used.

[0128] DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: NIS5P00; Serum matrix: Human serum AB male, HIV tested, from BIOWEST SAS, Nuaille, France, Lot: S00SS10015) with 4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) and then incubated for 2 min in the upper cavity of the ID-Card Coombs Anti-IgG (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 972264 48 01) at room temperature. Subsequently, 50 pl of the 0.8% panel cell #1 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025013) was added and incubated with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) for 15 min at 37°C. Anti-CD38 antibody-free serum (AB serum) served as a control for assessing a negative agglutination reaction (column #4).For the post-block procedure with daratumumab, 25 pl of the daratumumab-containing AB serum (daratumumab: 750 pg / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B 19; serum matrix: human serum AB male, HIV tested, from BIOWEST SAS, Nuaille, France, Lot: S00SS10015) were mixed with 50 gl of the 0.8% panel cell #5 (Panocell 10, IMMUCOR INC, Norcross, USA, Lot: 24120) in the upper cavity of the ID-Card LISS / Coombs anti-IgG + C3d (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 026586 24 15). The RBC / serum mix was incubated for 30 seconds. Subsequently, the RBC / serum mix was mixed with 4 gl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) and incubated for 15 min at 37°C. Anti-CD38 antibody-free AB serum served as a control for assessing a negative agglutination reaction (column #7).

[0129] Subsequently, the ID cards of all trials were centrifuged in the ID centrifuge 24S (DiaMed GmbH, Gressier FR, Switzerland) and the agglutination results were read within 30 minutes (see Fig.

[0130] 12). A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete inhibition of the binding of the interfering anti-CD38 antibody (daratumumab) to the RBCs. In conclusion, it can be stated that daratumumab-mediated interference in the IAT could be completely eliminated using the pre-block, co-block, and post-block techniques (see Fig. 12).

[0131] Example 13: Concentration-dependent elimination of daratumum-ab-mediated interference in co- and post-block methods

[0132] Sample preparation: For the experiments, a 0.8% cell suspension was prepared from panel cells #5 and #6 (Panocell 10, IMMUCOR INC, Norcross, USA, Lot: 24120) using a dilution medium (ID-Diluent 2; Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland; Lot: 964053 00 41). For the co-block procedure with daratumumab, 25 gl of the daratumumab-containing AB serum (daratumumab: 750 g / ml; Stock:

[0133] DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B 19; Serum matrix: Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS 10015) with 4 gl of Blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) of various dilutions (1:100, 1:600, 1:1200, 1:1800) in the upper cavity of the ID-Card LISS / Coombs anti-IgG + C3d (Bio-Rad (DiaMed GmbH), Gressier FR, Switzerland, Lot: 026586 24 15) and then incubated for 2 min at room temperature.

[0134] Subsequently, 50 gl of the 0.8% panel cell #6 (Panocell 10, IMMUCOR INC, Norcross, USA, Lot: 24120) was added to the upper cavity of the ID card, which was incubated together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) for 15 min at 37°C. Anti-CD38 antibody-free serum from the AB group served as a control for assessing a negative agglutination reaction (column #1).

[0135] For the post-block procedure with daratumumab, 25 gl of the daratumumab-containing AB serum (daratumumab: 750 g / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B 19; Serum matrix: Human serum AB male, HIV tested, from BIOWEST SAS, Nuaille, France, Lot: S00SS10015) were mixed with 50 gl of the 0.8% panel cell #5 (Panocell 10, IMMUCOR INC, Norcross, USA, Lot: 24120) in the upper cavity of the ID-Card LISS / Coombs anti-IgG + C3d (Bio-Rad (DiaMed GmbH), Gressier FR, Switzerland, Lot: 026586 24 15). The RBC / serum mix was pre-incubated for 30 seconds. Subsequently, the RBC / serum mix was mixed with 4 gl of Blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) at various dilutions (1:100, 1:600, 1:1200, 1:1800) and incubated for 15 min at 37°C. Anti-CD38 antibody-free serum from the blood served as a control for assessing a negative agglutination reaction (column #7).Subsequently, the ID cards from all trials were centrifuged in the ID-Centrifuge 24S (DiaMed GmbH, Gressier FR, Switzerland), and the agglutination results were read within 30 minutes. A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete inhibition of the binding of the interfering anti-CD38 antibody to the RBCs (see Fig. 13). In conclusion, it can be stated that in both the co-block and post-block approaches, blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) successfully eliminated daratumumab-mediated interference on ID cards (Bio-Rad (DiaMed GmbH), Gressier FR, Switzerland) in a concentration-dependent manner (see Fig. 13).

[0136] Example 14: Dose-dependent elimination of isatuximab-mediated interference using the co-block method

[0137] Sample preparation: For the experiments, a 0.8% cell suspension was prepared from the panel cells #5 (Data-Cyte Plus 3%, Grifols, Lot: 612025019) with a dilution medium (ID-Diluent 2; Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 964053 00 41).

[0138] For the co-block procedure with isatuximab, 25 pl of the isatuximab-containing AB serum (isatuximab: 1000 pg / ml; Stock: SARCLISA 20 mg / ml from Sanofi, Lot: 3F005A; Serum matrix: Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS10015) were mixed with 10 pl, 5 pl, 2.5 pl or 1 pl of blocker #2 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 4) in the upper cavity of the ID-Card anti-IgG (Bio-Rad (DiaMed GmbH), Gressier FR, Switzerland; Lot: 972264 48 01) and then incubated for 2 min at room temperature. Subsequently, 50 pl of the 0.8% panel cell #5 (Data-Cyte Plus 3%, Grifols, Lot: 612025019) was added to the upper cavity of the ID card, which was incubated together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 4) for 15 min at 37°C. AB serum without anti-CD38 antibody served as a control for the assessment of a negative agglutination reaction (column #1).

[0139] Subsequently, the ID cards from all trials were centrifuged in the ID-Centrifuge 24S (DiaMed GmbH, Gressier FR, Switzerland), and the agglutination results were read within 30 minutes. A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete inhibition of the binding of the interfering anti-CD38 antibody (isatuximab) to the RBCs (see Fig. 14). In conclusion, it can be stated that in the co-block procedure, blocker #2 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 4) was able to eliminate isatuximab-mediated interference on ID cards (Bio-Rad (DiaMed GmbH), Gressier FR, Switzerland) in a dose-dependent manner (see Fig. 14).

[0140] Example 15: Concentration-dependent elimination of daratumum-ab-mediated antibodies using the co-block method

[0141] Sample preparation: For the execution of the experiments on the Anti-IgG Ortho BioVue System (Ortho-Clinical Diagnostics, Pencoed, UK, Lot: IGC161H), a 4% cell suspension was prepared from panel cell #2 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) with the dilution medium (0.85% NaCl with 0.1% NaNs).

[0142] For the experiments on DG Gel Coombs Cards (Diagnostic Grifols, SA, Barcelona, ​​Spain, Lot: 25072.01), a 0.8% cell suspension was prepared from panel cell #2 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) using the dilution medium (DG Gel Sol, Diagnostic Grifols, SA, Barcelona, ​​Spain, Lot: 24019). For the co-block procedure with daratumumab on Anti-IgG Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK, Lot: IGC161H), 40 gl of the daratumumab-containing AB serum (daratumumab: 750 g / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany) were used. Germany, Lot: PCS5B19; Serum matrix: Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS10015) with 6.4 gl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No.49) various dilutions (1:20, 1:40, 1:80, 1:160, 1:320, 1:640) were added and then incubated for 2 min at room temperature in the upper cavity of the Anti-IgG Ortho BioVue System cassette. Subsequently, 10 gl of the 4% cell suspension (panel cell #2, Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015 in 0.85% NaCl with 0.1% NaNs) was added, which, together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49), was incubated for 15 min at 37°C. AB serum without anti-CD38 antibody served as a control for the assessment of a negative agglutination reaction (column #1).

[0143] For the co-block procedure with daratumumab on DG Gel Coombs Cards (Diagnostic Grifols, SA, Barcelona, ​​Spain, Lot: 25072.01), 25 gl of daratumumab-containing AB serum (daratumumab: 750 g / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B19; Serum matrix: Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS10015) were mixed with 4 gl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) of various dilutions (1:20, 1:40, 1:80, 1:160, 1:320, 1:640) and then incubated for 2 min at room temperature in the upper The cavity of the DG Gel Coombs Card was incubated. Subsequently, 50 gl of the 0.8% panel cell #2 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) was added using DG Gel Sol (Diagnostic Grifols, SA)., Barcelona, ​​Spain, Lot: 24019) which was diluted together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) for 15 min at 37°C. AB serum without anti-CD38 antibody served as a control for the assessment of a negative agglutination reaction (column #9).

[0144] The respective gel card was then centrifuged in a suitable centrifuge, and the agglutination results were read within 30 minutes. A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete blockade of the binding of the present anti-CD38 antibody (daratumumab) to the RBCs. In conclusion, it can be stated that daratumumab-mediated interference could be eliminated in a concentration-dependent manner using the co-block method on both Anti-IgG Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK) and DG Gel Coombs cards (Diagnostic Grifols, SA, Barcelona, ​​Spain) (see Fig. 15).

[0145] Example 16: Determination of irregular anti-K antibodies in daratumumab-containing sera using the co-block method

[0146] Sample preparation: For the experiments on the ID-Card LISS / Coombs anti-IgG + C3d (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 026586 24 15), samples were taken from the stocks of panel cells #1 - #11 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) and diluted with a diluent medium (ID-Diluent 2 from Bio-Rad (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot:

[0147] 964053 00 41) prepared a 0.8% cell suspension in each panel. Panel cells #2 and #8 represented RBCs expressing the Kell blood group antigen on their surface, while all other RBCs examined did not express the Kell blood group antigen.

[0148] For the experiments on DG Gel Coombs Cards (Diagnostic Grifols, SA, Barcelona, ​​Spain, Lot: 25072.01), a 0.8% cell suspension was prepared from the stocks of panel cells #1–#11 (Data-Cyte Plus 3%, Medion GrifolsDiagnostics AG, Düdingen, Switzerland, Lot: 612025015) using the dilution medium (DG Gel Sol; Diagnostic Grifols, SA, Barcelona, ​​Spain, Lot: 24019). Panel cells #2 and #8 represented red blood cells (RBCs) expressing the Kell antigen on their surface, while all other RBCs examined did not express the Kell antigen.

[0149] For conducting the experiments on Anti-IgG Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK, Lot:

[0150] IGC161H) was obtained from the stocks of panel cells #1 - #11 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot:

[0151] 612025015) a 4% cell suspension was prepared with the dilution medium (0.85% NaCl with 0.1% NaNs). Panel cells #2 and #8 represented RBCs expressing the Kell blood group antigen on their surface, while all other RBCs examined did not express the Kell blood group antigen.

[0152] For the co-block procedure on ID-Card LISS / Coombs anti-IgG + C3d (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 026586 24 15), 25 pl of daratumumab-containing serum (daratumumab: 750 pg / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B19; serum matrix (without irregular anti-BG antibodies): Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS10015; serum matrix anti-Kell: CE-Immundiagnostika GmbH, Neckargemünd, Germany, Lot: MKM222-1) were mixed with 4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) and then for Incubated for 2 minutes at room temperature in the upper cavity of the gel card.Subsequently, 50 pl of the respective 0.8% cell suspension (#1 - #11, Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) was added to the corresponding column, which was incubated together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) for 15 min at 37°C.

[0153] For the co-block procedure on DG Gel Coombs Cards (Diagnostic Grifols, SA, Barcelona, ​​Spain, Lot: 25072.01), 25 pl of daratumumab-containing serum (daratumumab: 750 pg / ml, Stock: ) were used.

[0154] DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B 19; Serum matrix (without irregular anti-BG antibodies):

[0155] Human Serum AB Male, HIV tested, Biowest SAS, Nuaille, France, Lot: S00SS10015; Serum matrix anti-Kell: CE-Immundiagnostika GmbH, Neckargemünd, Germany, Lot: MKM222-1) was mixed with 4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) and then incubated for 2 min at room temperature in the upper cavity of the gel card. Subsequently, 50 pl of the respective 0.8% cell suspension (#1 - #11, Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: ) was added.

[0156] 612025015) into the corresponding column, which was incubated together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) for 15 min at 37°C.

[0157] For the co-block procedure on anti-IgG Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK, Lot: IGC161H), 40 pl of daratumumab-containing serum (daratumumab: 750 pg / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B19; serum matrix (without irregular anti-BG antibodies): Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS10015; serum matrix anti-Kell: CE-Immundiagnostika GmbH, Neckargemünd, Germany, Lot: MKM222-1) were mixed with 6.4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) and then incubated for 2 min at room temperature in the upper cavity of the Gel card incubated.Subsequently, 10 pl of the respective 4% cell suspension (#1 - #11, Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) was added, which was incubated together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) for 15 min at 37°C.

[0158] The respective gel card was then centrifuged in a suitable gel card centrifuge, and the agglutination results were read within 30 minutes. A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete blockage of the binding of the present anti-CD38 antibody (daratumumab) to the RBCs. In conclusion, it can be stated that the daratumumab-mediated interferences were eliminated via the co-blocking procedure with blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49), and at the same time, anti-Kell antibodies were correctly detected in the antibody screening test in all gel card systems examined (see Fig. 16).

[0159] Example 17: Comparison of the co-block and post-block methods for the determination of irregular anti-Kell or anti-Jk(a) antibodies

[0160] Sample preparation: For the experiments on anti-IgG, -C3d; polyspecific Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK) and anti-IgG Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK), a 0.8% cell suspension was prepared from the respective K+ / K- or Jk(a+) / Jk(a-)- panel cells (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) with the dilution medium (0.85% NaCl with 0.1% NaNs).

[0161] For the co-block method against anti-IgG, -C3d; Polyspecific Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK, Lot: AHC399F) and anti-IgG Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK, Lot: IGC161H) were used to fill the corresponding column cavities with 40 pl of (daratumumab-containing) serum (daratumumab: 750 pg / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B 19; serum matrix (without irregular anti-BG antibodies): Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS10015; anti-Kell serum: CE-Immundiagnostika GmbH, Neckargemünd, Germany, Lot: MKM222-1) and with / without Irregular blood group antibodies of various specificities (anti-Kell; anti-Jk(a)) mixed with 6.4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) in column #5, #6, #11, #12 and then incubated for 2 min at room temperature.An AB serum (column 1 and column 7: without MAB) served as a negative control in each case. Column 2 and column 8 served as positive controls for anti-Kell and anti-Jk(a)-mediated agglutination, respectively. The BG-negative cell line in column 4 and column 10 served as positive controls for daratumumab-mediated agglutination. Anti-Kell and anti-Jk(a)-mediated agglutination, including daratumumab, was performed in columns 3-6 and columns 9-12, respectively. After incubation, 50 µl of the respective 0.8% cell suspension was added and incubated with the serum / blocker mix for 15 min at 37°C.

[0162] For the post-block assay for anti-IgG, -C3d; Polyspecific Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK, Lot: AHC399F) and anti-IgG Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK, Lot: IGC161H) were used to fill the corresponding column cavities with 40 pl of (daratumumab-containing) serum (daratumumab: 750 pg / ml; DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B 19; serum matrix (without irregular anti-BG antibodies): Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS 10015; anti-Kell serum: CE-Immundiagnostika GmbH, Neckargemünd, Germany, Lot: MKM222-1) as well as with / without irregular Blood group antibodies of various specificities (anti-Kell; anti-Jk(a)) were mixed with 50 pl of the respective 0.8% cell suspension and after approximately 30-60 minutes, 6.4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) were added to columns #17, #18, #23, #24.Incubation was performed for 15 minutes at 37°C. An AB serum (column 13 or column 19: without MAB) served as a negative control. Column 14 or column 20 served as a positive control for anti-Kell or anti-Jk(a)-mediated agglutination. The respective BG-negative cell line in column 16 or column 22 served as a positive control for daratumumab-mediated agglutination. Anti-Kell or anti-Jk(a)-mediated agglutination including daratumumab was performed in columns 15-18 or columns 21-24.

[0163] Subsequently, the anti-IgG, -C3d polyspecific Ortho BioVue System cassettes and the anti-IgG Ortho BioVue System cassettes were centrifuged in a BioVue® card centrifuge, and the agglutination results were read within 30 minutes. A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete blockage of the binding of the present anti-CD38 antibody (daratumumab) to the RBCs. In conclusion, it can be stated that in both the co-block and post-block approaches, blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) inhibited daratumumab-mediated interference on anti-IgG, -C3d; polyspecific Ortho BioVue System cassettes and the anti-IgG Ortho BioVue System cassettes were able to eliminate without interfering with the detection of irregular blood group antibodies (anti-Kell and anti-Jk(a)) (see Fig. 17).

[0164] Example 18: Co-block method for the determination of irregular anti-Kell or anti-Jk(a) antibodies in isatuximab-containing sera

[0165] Sample preparation: For the experiments, samples were taken from the respective K+ / K- or Jk(a+) / Jk(a-) panel cells (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot:

[0166] 612025019) a 0.8% cell suspension with the dilution medium (ID-Diluent 2; Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 964053 00 41).

[0167] For the co-block procedure with isatuximab on ID cards Coombs Anti-IgG (Bio-Rad (DiaMed GmbH), Gressier FR, Switzerland; Lot: 972264 48 01), the corresponding column cavities were filled with 25 pl of the (isatuximab-containing) serum (Isatuximab: 1000 pg / ml; Stock: SARCLISA 20 mg / ml from Sanofi, Lot: 3F005A; Serum matrix: Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS10015; anti-Kell serum: CE-Immundiagnostika GmbH, Neckargemünd, Germany, Lot: MKM222-1; anti-Jk(a) serum: CE-Immundiagnostika GmbH, Neckargemünd, Germany, Lot: MJkaM237-l) and with / without irregular blood group antibodies of various specificities (anti-Kell; anti-Jk(a)) with 3 pl of blocker #2 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 4) in columns #5, #6, #11, #12 and then incubated for 2 min at room temperature. An AB serum (column 1 and column 7: without MAB) served as a negative control in each case.Column 2 and column 8 served as positive controls for anti-Kell and anti-Jk(a)-mediated agglutination, respectively. The respective K or JK(a) cell line in column 4 and column 10 served as positive controls for isatuximab-mediated agglutination. Anti-Kell and anti-Jk(a)-mediated agglutination, including daratumumab, was performed in columns 3-6 and columns 9-12, respectively. After incubation, 50 pl of the respective 0.8% cell suspension was added and incubated with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 4) for 15 min at 37°C.

[0168] Subsequently, the ID cards from all trials were centrifuged in the ID-Centrifuge 24S (DiaMed GmbH, Gressier FR, Switzerland), and the agglutination results were read within 30 minutes. A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete inhibition of the binding of the interfering anti-CD38 antibody (isatuximab) to the RBCs. In conclusion, the application of the Co-Block method Blocker #2 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 4) eliminated isatuximab-mediated interference on ID cards Coombs anti-IgG without interfering with the detection of irregular blood group antibodies against anti-Kell and anti-Jk(a) (see Fig. 18).

[0169] Example 19: Elimination of daratumumab-mediated interference in authentic patient plasma using the co-block method

[0170] Sample preparation: For the execution of the experiments on the ID-Card LISS / Coombs Anti-IgG + C3d (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 026586 24 15), a 0.8% cell suspension was prepared from panel cell #6 (Panocell 10, IMMUCOR INC, Norcross, USA, Lot: 20090) with a dilution medium (ID-Diluent 2; Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 964053 00 41).

[0171] For the experiments on DG Gel Coombs Cards (Diagnostic Grifols, SA, Barcelona, ​​Spain, Lot: 25072.01), a 0.8% cell suspension was prepared from panel cell #8 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) using the dilution medium (DG Gel Sol; Diagnostic Grifols, SA, Barcelona, ​​Spain, Lot: 24019).

[0172] For conducting the experiments on Anti-IgG Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK), Lot:

[0173] IGC161H) was used to prepare a 0.8% cell suspension with the dilution medium (0.85% NaCl with 0.1% NaNs) from panel cell #8 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015).

[0174] For the co-block procedure with daratumumab on ID-Card LISS / Coombs anti-IgG + C3d (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 026586 24 15) and DG Gel Coombs Cards (DiagnosticGrifols, SA, Barcelona, ​​Spain, Lot: 25072.01), 25 pl of the respective daratumumab-containing patient plasma were mixed with 4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) and then incubated for 2 min in the upper cavity of the gel card at room temperature. Subsequently, 50 pl of the respective 0.8% RBC suspension, in this case RBC #6 or RBC #8, was added to the corresponding column cavity, which was incubated together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) for 15 min at 37°C.

[0175] For the co-block procedure with daratumumab on anti-IgG Ortho BioVue System cassettes (Ortho-Clinical Diagnostics, Pencoed, UK, Lot: IGC161H), 40 pl of the respective daratumumab-containing patient plasma were mixed with 6.4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) and then incubated for 2 min at room temperature in the upper cavity of the gel card. Subsequently, 50 pl of the 0.8% RBC suspension (RBC #8) was added, which, together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49), was incubated for 15 min at 37°C.

[0176] The respective gel card was then centrifuged in a suitable centrifuge, and the agglutination results were read within 30 minutes. A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete blocking of the binding of the present anti-CD38 antibody (daratumumab). In conclusion, it can be stated that, via co-incubation with blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49), daratumumab-mediated interferences could be eliminated in all patient sera examined on all gel card systems used (see Fig. 19). Example 20: Neutralization of daratumumab-mediated interference and simultaneous detection of irregular anti-Kp(a) antibodies using the standardized (2 min) and extended (60 min) co-incubation protocols.

[0177] Sample preparation: For the experiments, a 0.8% cell suspension was prepared from panel cells #5 and #2 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) with a dilution medium (ID-Diluent 2, Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 964053 00 41).

[0178] For the co-block procedure, 25 pl of the respective (daratumumab-containing) serum (daratumumab: 750 pg / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B 19; serum matrix (without irregular anti-BG antibodies): Human Serum AB Male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS10015; anti-Kp(a) serum: CE-Immundiagnostika GmbH, Neckargemünd, Germany, Lot: MKpaM058-l) were combined with 4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49), which was incubated in the upper cavity of the ID-Card LISS / Coombs anti-IgG + for two different co-incubation periods (2 min and 60 min, respectively). Bio-Rad C3d (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 026586 24 15) was co-incubated with the serum. Subsequently, 50 pl of the diluted panel cell #2 or #5 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 026586 24 15) was added.

[0179] 612025015), which was incubated together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) for 15 min at 37°C.

[0180] Subsequently, the ID cards of all trials were centrifuged in a 24S ID centrifuge, and the agglutination results were read within 30 minutes. A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete inhibition of the binding of the interfering anti-CD38 antibody (daratumumab) to the RBCs. In conclusion, it can be stated that the daratumumab-mediated interference in the IAT could be completely eliminated via the co-blocking procedure, regardless of the investigated co-incubation time of blocker #3 with the serum (see Fig. 20).

[0181] Example 21: Initial stress tests to investigate the functional limitations of blocker #3 at elevated storage temperatures

[0182] Sample preparation: Prior to performing the lATs, aliquots of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) were exposed to a wide variety of storage conditions (storage conditions: 24h at +20°C; 48h at +20°C; 24h at +35°C; 48h at +35°C; 24h at +45°C; 48h at +45°C; 24h at +65°C; 48h at +65°C). For the experiments, a 0.8% cell suspension was prepared from panel cells #2 and #3 (Data-Cyte Plus 3%, Medion Grifols Diagnostics AG, Düdingen, Switzerland, Lot: 612025015) with a dilution medium (ID-Diluent 2 from Bio-Rad (Bio-Rad (DiaMed GmbH), Cressier FR, Switzerland, Lot: 964053 00 41).

[0183] For the co-block procedure with daratumumab on ID-Card LISS / Coombs Anti-IgG + C3d (Bio-Rad (DiaMed GmbH, Cressier FR, Switzerland, Lot: 026586 24 15)), the corresponding column cavities were filled with 25 pl of the (daratumumab-containing) serum with or without irregular anti-Fy(a) antibody (daratumumab: 750 pg / ml; Stock: DARZALEX 20 mg / ml from Janssen-Cilag GmbH, Neuss, Germany, Lot: PCS5B 19; serum matrix (without irregular anti-BG antibody):

[0184] Human serum AB male, HIV tested, from Biowest SAS, Nuaille, France, Lot: S00SS 10015; serum matrix anti-Fy(a) serum: BAG Diagnostics GmbH, Lieh, Germany, Lot: 270324) was mixed with 4 pl of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) and then incubated for 2 min at room temperature. An AB serum (column #1, #7, #13, #19, #25, #31, #37, #43) served as a negative control for the IATs. An anti-Fy(a) serum (column #2, #8, #14, #20, #26, #32, #38, #44) served as a positive control for anti-Fy(a)-mediated agglutination. A daratumumab-containing anti-Fy(a) serum in combination with an Fy(a) cell line served as a positive control for daratumumab-mediated agglutination (columns #4, #10, #16, #22, #28, #34, #40, #46). The anti-Fy(a) / daratumumab-mediated agglutination reaction was performed in columns #3-6, #9-12, #15-18, #21-24, #27-30, #33-36, #39-42, #45-48.After preparation of the blocker / serum mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49), 50 pl of the respective 0.8% cell suspension # 2 or #3 was added, which together with the serum / blocker mix (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) was incubated for 15 min at 37°C.

[0185] Subsequently, the ID cards of all trials were centrifuged in a 24S ID centrifuge, and the agglutination results were read within 30 minutes. A completely negative result (a flat pellet of cells at the bottom of the microcolumn) indicated complete inhibition of the interfering anti-CD38 antibody (daratumumab) binding to the RBCs. In conclusion, storage conditions influenced the stability of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49), with only one instance of blocker #3 (containing a polypeptide with an amino acid sequence according to SEQ ID No. 49) being observed at a storage temperature of +65°C, regardless of the storage duration.All other storage conditions tested had no negative impact on the elimination of daratumumab-mediated agglutination and the detection of irregular anti-Fy(a) antibodies (see Fig. 21A and Fig. 21B).

[0186] Example 22: Tabular overview of the polypeptides according to the invention. In the following Table 1, the amino acid sequences of the polypeptides according to the invention are shown according to SEQ ID No. 1 to SEQ ID No. 58.

[0187] Table 1

[0188] SEQ amino acid sequence

[0189] ID

[0190] No.

[0191] 1 QVQLQESGGGLVQPGGSLRLSCGATG11FS1NDMGWYRQAPGKQREL VAE1TRGGSTNYADSVKGRFT1SRDNAKNTVYLQMNSLNPEDTAVY YCNAAHTFSGSFWGQGTQVTVSS

[0192] 2 QVQLQESGGGLVQPGGSLRLSCGATG11FS1NDMGWYRQAPGKQREL VAE1TRGGSTNYADSVKGRFT1SRDNAKNTVYLQMNSLNPEDTAVY YCNAAHTFSGSFWGQGTQVTVSS

[0193] 3 QVKLQESGGGLVQPGGSLRLSCGATG11FS1NDMGWYRQAPGKQREL VAE1TRGGSTNYADSVKGRFT1SRDNAKNTVYLQMNSLNPEDTAVY YCNAAHTFSGSFWGQGTQVTVSS

[0194] 4 QVQLQESGGGLVQPGGSLRLSCAASG11LR1YDMGWYRQAPGKQREL VAA1TSRGSTNYADSVKGRFT1SRDNAENTVSLQMNSLKPGDTAVY YCNADHTFAGVYWGQGTQVTVSS

[0195] 5 QVQLQESGGGLVQPGGSLRLSCAASG11LR1YDMGWYRQAPGKQREL VAA1TSGGRTNYADSVKGRFT1SRDNAENTVSLQMNSLKSGDTAVY YCNADHTFAGVYWGQGTQVTVSS

[0196] 6 QVQLQESGGGLVQPGGSLRLSCAASG11LR1YDMGWYRQAPGKQREL VAE1TSGGSTNYADSVKGRFT1SRDNAENTVSLQMNSLKPGDTAVYY CNAHHTFAGAYWGQGTQVTVSS

[0197] 7 QVQLQESGGGLVQPGGSLRLSCAASG11LRLYDMGWYRQTPGKQRE LVAE1TSGGSTNYADSVKGRFT1SRDNAENTVSLQMNSLKSGDTAVY YCNADHTFAGVYWGQGTQVTVSS

[0198] 8 QVQLQESGGGLVQPGGSLRLSCAASGSTLRLYDMGWYRQADGKQR ELVAE1TSGGSTNYANSVKGRFT1SRDNAENTVSLQMNSLKPEDTAV YYCNAEHTFMGAYWGQGTQVTVSS

[0199] 9 QVQLQESGGGLVQAGGSLRLSCAASG11LR1YDMGWYRQAPGKQREL VAA1TSGGSTNYADSVKGRFT1SRDNAKSMVYLQMNSLKPEDTAVY YCNAWHVFRGNYWGQGTQVTVSS

[0200] 10 QVQLQESGGGLVQAGGSLTLSCAASRNTDS1E1MGWYRQAPGKQRE WVAT1NTGGNTGYADSVKGRFA1SRDNAKNTVYLQMHSLKPEDTA VYYCTAV1RTYSTYWGQGTQVTVSS

[0201] 11 QVQLQESGGGLVQPGGSLRLSCVASER1FSRNTMGWYRQAPGKQRE LVAR1ASLG1TNYADSVKGRFT1SSDNAKNTVYLQMNNLKPEDTAVY YCNYWHYAAGRDYWGQGTQVTVSS

[0202] 12 EVQLVESGGGLVQAGGSLRLSCVASER1FSRNTMGWYRQAPGKQRE LVAR1ASLG1TNYADSVKGRFT1SSDNAKNTVYLQMNNLKPEDTAVY YCNYWHYAAGRDYWGQGTQVTVSS

[0203]

[0204] 13 QVQLQESGGGLVQPGGSLRLSCVGSER1FSRNTMGWYRQAPGKQRELVGYVGSMGITNYADSVKGRFTISSDNAKNTVYLQMNNLKPEDTAV YYCNYWHYAAGRDYWGQGTQVTVSS QVKLQESGGGLVQPGGSLRLSCVASERIFSRNTMGWYRQAPGKQRE LVAYITSLGIANYADSVKGRFTISSDNAKNTVYLQMNNLKSEDTAVY NCNYWHYAAGRDYWGQGTQVTVSS DVQLQASGGGSVQAGGSLTLSCRYSGGFVFRVSSMGWYRQAPGNQR ELIATITVGGKTNYKDSVQGRFIISRNDTTVTLQMNRLQPEDTAVYY CNLASTAVGADTWGQGTRVTVSS DVQLQESGGGSVQAGGSLTLSCTASGLLFRLASMGWYRQAPGKERE LIATITVGGKTNYKDSVQGRFIITRDNTGDNTKSTVTLQMNRLKPED TAVYYCNTASPAVGADTWGQGTRVTVSS QVKLQESGGGLVQPGGSLRLSCVASGSISSIITMGWYRQAPGKQREF VARVIIGGSTGYADSVKGRFTISRDNTKNTVYLEMNSLKPEDTAVYY CNAGNPGTSYHYWGQGTQVTVSS QVQLQESGGGLVQPGGSLRLSCVASGSISNIITMGWYRQAPGKQREF VARIIIGGSTGYSDSVKGRFTISRDNTGNTWYLQMNSLKPEDTAVYY CNAGNPGTRYIYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGRTFSSVAMGWFRQAPGKERE FVATINWSGGRTTYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTA VYYCAADRFSVVAVEYDYWGQGTQVTVSS EVQLVESGGGLVQAGGSLRLSCAASGRTFSSVAMGWFRQAPGRERE FVATINWSGGRTTYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTA VYYCAADRFSVVAVEYDYWGQGTQVTVSSQVQLQESGGGLVQAGGSLRLSCAISGRTFIADMGWFRQAPGKEREF QAGITWFGGSTYYADSVKGRFTISRDNTKNTLYLQMNSLKPEDTAI YYCAAGLKRIGDQREADYWGQGTQVTVSS EVQLVESGGGLVQAGGSLRLSCAISGRTFIADMGWFRQAPGKEREF QAGITWFGGSTYYADSVKGRFTISRDNTKNTLYLQMNSLKPEDTAI YYCAAGLKRIGDQREADYWGQGTQVTVSS QVQLQESGGGLVQAGGSVRLSCAISGRTFINDMGWFRQAPGKEREF QAGITWFGGSTYYADSVKGRFTISRDNTKNTLYLQMNSLKPEDTAI YYCAAGLKRIGDQREADYWGQGTQVTVSS DVQLQASGGGLVQAGGSLRLSCAGSGRTFTNYDMGWFRQAPGKER RFVAAISGSGGSARYAVPVKGRFTITRDNAKNTVYLQMSSLKPEDTA VYYCAADRFVVAAGTHDLDYWGQGTQVTVSS DVQLQESGGGLVQAGGSLRLSCAGSGRTFTNYDMGWFRQAPGKER RFVAAISGSGGSARYAVPVKGRFTITRDNAKNTVYLQMSSLKPEDTA VYYCAADRFVVAAGTHDLDYWGQGTQVTVSS DVQLQASGGGLVQAGGSLRLSCAGSGRSFSSYAMGWFRQAPGKARE IVATITRSGGSTNYAVPVKGRFTISRDNAKNTVYLQMSSLKPEDTAV YYCAADRFAVASGTHDLDYWGQGTQVTVSS DVQLQASGGGLVQAGGSLRLSCAGSGRTFSSYAMGWFRQAPGKERE FVASISWSGSRTNYGVPVKGRFTISRDNAKNTVYLQMSSLKPEDTA VYYCAADRFAVAIGTHDLDYWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGRTDTRYTMGWFRQAPGKER EFVSGITWNGGTSYADSVKGRFTISRDNGKNSVYLQMNSSLKPEDTA

[0205]

[0206] VYYCAADRFTLVPTTSDLDYWGQGTQVTVSSQVQLQESGGRLVQPGGSLRLSCAASGRTLSNYAMAWFRQGPGKERE FVASISASDSTLYADFVKGRFTISRDNAKNTVYLQMDSLKPEDTTVY YCAARFWIGVRAPAEYNYWGQGTQVTVSS QVKLQESGGRLVQPGGSLRLSCAASGRTLSNYAMAWFRQGPGKERE FVASISASDSTLYADFVKGRFTISRDNAKNTVYLQMDSLKPEDTTVY YCAARFWIGVRAPAEYNYWGQGTQVTVSS QVKLQESGGRLVQPGGSLRLSCAASGRTFSNYAMAWFRQGPGKERE FVAAISAADSTLYADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YYCAARWWIAVRAPAEYNYWGQGTQVTVSS QVQLQESGGRLVQPGGSLRLSCAASGRTFSNYAMAWFRQGPGKERE FVAAISAADSTLYADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAV YYCAARWWIAVRAPAEYNYWGQGTQVTVSS DVQLQASGGGLVQAGGSLRLSCAPSGGTFDAYDMGWYREAPGKKR QFVAAVRSGGGTTRYADSVKGRFTISRDDAKDTVFLQMNSLKPEDT ALYYCAADRWGLFSLSIATPTHWGQGTQVTVSS DVQLQASGGGSVQEGESLKLSCVGSGRTFSDWAMGWYRQAPGKDR EFVAAVSGAGRGGKPSYANSVKGRFTVSRDNAKNTVYLQMDNLKP EDTAVYYCAADRLVLVALSIADPGFWGQGTRVTVSS DVQLQESGGGLVQAGGSLRLSCAASGVSFSRYTMGWYREVPGKERR EFVAAVRPSGDSTYYGNSVKGRFSISRDDDKNIVYLQMNSLKPEDTA VYYCAAGFPVLVALSIADPDYWGQGTQVTVST DVQLQASGGGLVQTGDSLTVSCAASGLSFTRYTMGWYREVPGKERREFVAAVKPAGDSAYYGASVKGRFTASRDNAKNTVTLQMNSLKPED TAIYYCAAGFPVLTALHIADPDYWGQGTQVTVSP DVQLQESGGGLVQAGGSLRLSCTGSGRTFRNYPMAWFRQAPGKER EFVAGITWVGASTLYADFAKGRFTISRDNAKNTVYLQMNSLKPEDT AVYSCAAGRGIVAGRIPAEYADWGQGTQVTVSS QVQLQESGGGLVQAGGSLRLSCAASGRTFVSFGMGWFRQAPGKERE FVAAINWRGSTTAYADSVKGRFTISRDVTKNTIYLQMNSLKPEDTAI YYCAEGRTASASTMIREYDSWGQGTQVTVSE DVQLQASGGGSVQAGGSLRLSCAASGLSFSDYAMGWFRQEPGKERR FVASIRSSGGTTYAESVKGRFTISRDNAKNTMYLQMNNLKPEDTAV YYCAADRLVITKLSIADPGYWGQGTQVTVSS VQLQASGGGLVQAGGSLRLSCTVSGFTSDDYTVGWFRQAPGLKREG LSCLSRRDGRFYHSNSVKGRFTMXSDDXKNTVYLQLDSLKPDXTAV XYCAACTSVVVLLAPNWYEYWGQGTQVTVSS VQLQASGGGLVQAGGSLRLSCTVSGFTSDDYTVGWFRQAPGLKREG LSCLSRRDGRFYHSNSVKGRFTMSSDDNKNTVYLQLDSLKPDDTAV YYCAATTSVVVLLAPNWYEYWGQGTQVTVSS EVQLVESGGGLVQAGESLRLSCAAGRTFTNLGMGWFRQAPGKEREF VAADTWSGTSTWYGDSVKGRFTISRDNAKNTVYLQMNNLKPEDTA VYYCAARLRGWITTRKPNEYDYWGQGTQVTVAS DVQLQESGGGLVQAGDSLRLSCEFSGRTFSGFAMGWFRQFPGKERE FVAAINWSGSDTDYSDSVKGRFTISRDNVKQMMYLAMNSLKPEDT AVYFCAEARSAGLGSSRRIEGYDKWGRGTQVTVSSDVQLQESGGGLVQAGDSLRLSCEFSGRTFSGFAMGWFRQFPGKERE

[0207]

[0208] FVAAINWSGSDTDYSDSVKGRFTISRDNVKQMMYLAMNSLKPEDTAVYFCAEARSAGLGSSRRIEGYDKWGRGTQVTVSS DVQLQASGGGLVQAGGSLRLSCEFSGRTFSGFAMGWFRQFPGKERE FVAAINWSGSSVDYSDSVKGRFTISRDNVKSTMYLVMNSLKPEDTA VYYCAEARSAELGSSRKIQGYDQWGRGTQVTVSS DVQLQASGGGLVQAGGSLRLSCEFSGRTFSGFAMGWFRQFPGKERE FVAAINWSGSSVDYSDSVKGRFTISRDNVKSTMYLVMNSLKPEDTA VYYCAEARSAELGSSRKIQGYDQWGRGTRVTVSS DVQLQESGGGLVQAGSSLRISCAVSGVRLDNYAMGWFRQAPGKERE SVAGISWSSGTLLYSDSVKGRFAISRDNAKNTVYLQMNSLKPEDTAV YYCAAQYQDRYYDEFTWKEKDMDYWGKGTLVTVSS DVQLQESGGGLVQAGDSLRLSCAASGPHFNNYAIGWFRQAPGKERE FVAGISWSSGSLLYSDSVKGRFTISRDNDKNTAYLQMNSLKPEDTAL YYCAAQYQERYYSDFSLKEKGMEYWGKGTLVTVSS DVQLQESGGGLVQAGDSLRLSCVGSGRRFDNYAMAWFRQAPGKER TFVAAISWSSGTTRYLDTVKGRFTISRDNAKSTVYLQMNSLKPEDT AVYYCAARYQPRYYDSGDMDGYEYEFWGQGTQVTVSS DVQLQESGGGLVQAGHSLRLSCVGSGSRFDNYAMGWFRQAPGKER EFVAAISWSSGTTRYLDTVKGRFTISRDNAKSTVYLQMNSLKPEDT AVYYCAARYQPRYYDSGDMDGYEYDNWGQGTQVTVSS DVQLQASGGGLVQAGGSQRLSCAASGHTFSSYSMAWFRQAPGKERE FVAANSMSGTKTAYSDSVKGRFTISRDIPKNTVYLQMNSLKPEDTA LYYCAAVDRSTGWDSWRDDPDQYDYWDQGTQVTVSSSGGGLVQAGGSQRLSCAASGHTFSSYSMAWFRQAPGKEREFVAANI MSGTNTYYADSVRGRFTMSRDIAKNTVYLQMNSLKEEDTALYYCA AADRFRGWATWRDDPDQYDYWDQGTQVTVSS DVQLQESGGGLVQAGGSLRLSCAASGFTFDDYVIGWFRQAPGKERE GVSSISNNNSTYYADSVKGRFTIASDNAKNTVYLQMNSLKPEDTAV YYCAADVTLNPFTGWNTRSGPMYRYEYDYWGQGTQVTVSS VQLQESGGGLVQAGGSLRLSCAASGFTFDDYVIGWFRQAPGKECEG VSSISNNNSTYYADSVKGRFTIASDNAKNTVYLQMNSLKPEDTAVYY CAADVTLNPFTGWNTRSGPMYRYEYDYWGQGTQVTVSS DVQLQASGGGLVQAGGSLRLSCAASEFTFDDYVIGWFRQAPGKERE GVSSISSDGSTYYADSVKGRFTISSDNAKNTVYLHMNSLKPEDTAVY YCAADVTLNPFTGWTTRSGPMYRYEYDYWGQGTQVTVSS VQLQASGGGLVQAGGSLRLSCAASGFTFDDYVIGWFRQSPGKEREG VSCISSAGSTYYADSVKGRFTISSDNAKNMVYLQMDNLKPEDTAVY YCAADVTLNPFTGWDTRSGPMYRYEYDYWGQGTQVTVSS DVQLQASGGGLVQAGGSLRLSCAASGFTFDDYVIGWFRQAPGKERE GVSSISSSGSIYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYF CAADVTLNPFTGWDTRSGPMYRYEYDYWGQGTQVTVSS DVQLQASGGGLVQAGGSLRLSCAASGFTFDDYVIGWFRQAPGKERE GVSSISSSGSIYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYF

[0209]

[0210] CAADVTLNPFTGWDTRSGPMYRYEYDYWGQGTQVTVSS

Claims

December 23, 2025 BAG Diagnostics GmbH G / BGD-006-WO 35423 Lieh Scw / kai Patent claims 1. Method for determining an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample, comprising the following steps: a) Providing a biological sample comprising erythrocytes expressing a transport protein (CD38); and b) Providing a polypeptide, in particular a single-domain polypeptide, which is specific for CD38 and comprises an amino acid sequence, wherein the amino acid sequence is selected from the group comprising: i) an amino acid sequence according to SEQ ID No. 1 to SEQ ID No. 58, which codes for an amino acid molecule; and ii) an amino acid sequence encoding an amino acid molecule that is at least 50% identical to the amino acid molecule encoded by the amino acid sequence from i); and iii) an amino acid sequence for a fragment of an amino acid molecule from i) or ii), wherein the fragment encodes a polypeptide; and c) contacting and incubating the biological sample with the polypeptide; and d) Determination of an anti-blood group antibody and / or an anti-CD38 antibody in the biological sample.

2. The method of claim 1, wherein a sample comprising an anti-CD38 antibody is provided, wherein the anti-CD38 antibody is specific for CD38, wherein the sample is brought into contact with and incubated with the biological sample in step c) before, during and / or after the contact and incubation of the biological sample with the polypeptide.

3. The method of claim 1 or 2, wherein the specified anti-blood group antibody is an antibody against an antigen from a blood group selected from ABO, MNS, P1PK, Rhesus, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Cartwright, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido / Rodgers, H-System, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, John Milton Hagen, I-System, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine, Kanno, Sid, CTL2, PEL, MAM, EMM, ABCC1, Er, CD36, ATP11C, MAL, Batty, Christiansen, Biles, Box, Torkildsen, Peters, Reid, Jensen, Livesay, Milne, Rasmussen, Jones, ABTI and LKE, is.

4. Method according to any one of claims 1 to 3, wherein the anti-CD38 antibody is present in a concentration of 0.001 pg / ml to 3500 pg / ml.

5. A method according to any one of claims 1 to 4, wherein the biological sample is human or animal blood, blood plasma and / or blood serum.

6. A method according to any one of claims 1 to 5, wherein at least two polypeptides are provided in step b).

7. A method according to any one of claims 1 to 6, wherein the polypeptide is added in step b) and / or in step c) in a concentration of The levels range from 0.01 pg / ml to 3000 pg / ml.

8. Method according to any one of claims 1 to 7, wherein the incubation in step c) is carried out at a temperature of 0°C to 42°C for a duration of 0 minutes to 60 minutes.

9. Method according to any one of claims 1 to 8, wherein the incubation in step c) comprises pre-incubating the biological sample, the polypeptide and / or the sample, wherein the pre-incubation is carried out at a temperature of 0°C to 42°C for a duration of 0 minutes to 120 minutes.

10. Device for determining an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample according to any one of claims 1 to 9, thereby marked, that the device has a receptacle for a container comprising a biological sample and a polypeptide, and that the device has an analysis, evaluation, and / or analysis unit for determining an anti-blood group antibody and / or the anti-CD38 antibody, wherein the biological sample comprises erythrocytes, the erythrocytes expressing a transport protein (CD38), and wherein the polypeptide is specific for CD38.

11. Kit for determining an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample according to any one of claims 1 to 9 comprising: a) a container comprising a biological sample comprising erythrocytes, wherein the erythrocytes express a transport protein (CD38), and a polypeptide, wherein the polypeptide is specific for CD38; and b) Instructions for carrying out the procedure.

12. Expression system for the production of a polypeptide comprising an amino acid sequence, wherein the polypeptide is specific for a transport protein (CD38) and wherein the amino acid sequence is selected from the group comprising: i) an amino acid sequence according to SEQ ID No. 1 to SEQ ID No. 58, which codes for an amino acid molecule; and ii) an amino acid sequence encoding an amino acid molecule that is at least 50% identical to the amino acid molecule encoded by the amino acid sequence from i); and iii) an amino acid sequence for a fragment of an amino acid molecule from i) or ii), wherein the fragment encodes a polypeptide, and where the expression system is either a eukaryotic expression system or a prokaryotic expression system.

13. Expression system according to claim 12, wherein the polypeptide has an affinity tag.

14. Expression system according to claim 12 or 13, wherein the polypeptide is a mono-specific, bi-specific, tri-specific or multi-specific single-domain polypeptide and / or wherein the polypeptide is monoclonal, polyclonal or a combination thereof.

15. Polypeptide manufactured according to any one of claims 12 to 14, wherein the polypeptide has a binding site and / or partially or completely blocks, inhibits and / or neutralizes the binding of an anti-CD38 antibody, in particular to erythrocytes, wherein the erythrocytes express a transport protein (CD38).

16. Polypeptide according to claim 15, wherein the anti-CD38 antibody is selected from daratumumab, felzartamab, CM313 and / or isatuximab.

17. Polypeptide according to claim 15 or 16 and / or a pharmaceutical composition comprising a polypeptide according to claim 15 or 16 and at least one further component for use in the determination of an anti-blood group antibody and / or an anti-CD38 antibody in a biological sample, wherein the biological sample comprises erythrocytes, wherein the erythrocytes express a transport protein (CD38).

18. Polypeptide according to claim 15 or 16 and / or a pharmaceutical composition comprising a polypeptide according to claim 15 or 16 and at least one further component for use in blocking, inhibiting and / or neutralizing an anti-CD38 antibody in a biological sample, wherein the biological sample comprises erythrocytes, wherein the erythrocytes express a transport protein (CD38).

19. Polypeptide according to claim 15 or 16 and / or a pharmaceutical composition comprising a polypeptide according to claim 15 or 16 and at least one further component for use in a therapeutic or non-therapeutic process for blocking, inhibiting and / or neutralizing an anti-CD38 antibody in a biological sample, wherein the biological sample comprises erythrocytes, the erythrocytes expressing a transport protein (CD38).

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