Analysis of anti-erythrocyte antibody in the presence of antibody directed against a surface-bound erythrocyte antigen
11131678 · 2021-09-28
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Abstract
An analytical process for detecting antibody in human blood serum, which antibody is directed against blood group antigen, wherein the serum contains an additional antibody directed against a surface antigen, by contacting the serum to be analysed with blood cell membranes, which naturally bear surface blood group antigens, followed by detection of agglutination which indicated the presence of antibody directed against at least one surface antigen of the blood cell membranes. The agglutination reaction caused by the additional antibody is prevented, allowing the detection of anti-blood group antibody.
Claims
1. Analytical process for detecting antibody specific for a blood group antigen in blood serum, comprising contacting the serum with blood cell membranes bearing surface antigens, and prior to contacting the serum with blood cell membranes, adding at least one binding peptide specifically binding to a first surface antigen of the blood cell membranes to the blood cell membranes, which binding peptide has no human Fc region; wherein the blood cell membranes are red blood cells or membrane fragments of red blood cells.
2. The analytical process according to claim 1 wherein the first antigen is not a blood group antigen.
3. The analytical process according to claim 2, wherein prior to contacting the serum with blood cell membranes, the method further comprises adding at least one binding peptide specifically binding to a blood group antigen to the blood cell membranes, which binding peptide has no human Fc region.
4. The analytical process according to claim 1 wherein the first antigen is a blood group antigen.
5. The analytical process according to claim 1, wherein the binding peptide is selected from the group consisting of F(ab).sub.2 fragments, Fab fragments, single-chain variable domain fragments (scFv), minibodies, diabodies, antibodies having a non-human Fc region, and proteins having at least one region forming at least one paratope specifically binding to the first antigen.
6. The analytical process according to claim 1, wherein the serum is contacted with the blood cell membranes in the presence of anti-human antibody.
7. The analytical process according to claim 1, wherein the blood cell membranes are bound to a synthetic carrier and wherein the antibody specific for a blood group antigen from blood serum is bound to the blood cell membranes and wherein the analytical process further comprises detecting said antibody by adding a labelled anti-human antibody.
8. The analytical process according to claim 1, further comprising detecting agglutination.
9. The analytical process according to claim 1, wherein the first antigen is CD38 and the antibody which is directed against a surface antigen of a blood cell is specific for CD38.
10. The analytical process according to claim 1, wherein the binding peptide is labelled with a detectable marker.
11. The analytical process according claim 1, wherein the serum originates from a patient who has been administered with an antibody directed against the first antigen.
12. The analytical process according to claim 1, wherein the binding peptide has the same paratope regions as an antibody having the same specificity as the binding peptide, which antibody is contained in the serum.
13. The analytical process according to claim 1, wherein the binding peptide is derived from the antibody having the same specificity.
Description
(1) The invention is now described in greater detail by way of examples with reference to the figures, which show in
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(5) In the examples, agglutination was determined to detect binding of antibody to the blood cell membranes, e.g. to detect binding of anti-blood group antibody of a serum to blood cell membranes bearing the specific blood group antigen.
Example 1: Analysis of Anti-Blood Group Antibodies in Serum
(6) As an example for an antibody directed against a first antigen present on all blood cell membranes, the therapeutic anti-CD38 antibody Daratumumab (DARA) was used in a 0.9% NaCl solution as a comparison (DARA only) or spiked into the serum prior to contacting with the blood cell membranes. As blood cell membranes, 60 μL erythrocytes were used in an agglutination assay using gel cards containing anti-human antibody (Coombs). The binding protein was the F(ab).sub.2 fragment generated from Daratumumab by pepsin digestion in 100 mM citric acid buffer for 60 min at 37° C. in a 40-fold molar excess of pepsin over Daratumumab. The digestion was stopped by adding Tris-buffered saline (TBS, pH 8.0). This binding protein is designated DARA-F(ab).sub.2. 100 μL of 0.3 μg/μL DARA-F(ab).sub.2 was added to 300 μL erythrocytes under shaking at room temperature as a pre-incubation, for comparative reactions, 100 μL TBS was used instead. For the agglutination reaction, 60 μL of these erythrocytes were incubated with 25 μL serum for 15 min at 37° C., layered onto the gel bed in the gel card wells, containing an anti-human antibody, and centrifuged according to the manufacturer's instructions.
(7) The erythrocytes were predetermined to be s positive for Cell-7 and s negative for Cell-11. The serum was human serum (obtained from Biolith, 1:2 dilution) known to contain anti-s antibody.
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(9) TABLE-US-00001 Well No. Cell serum Reaction 1 Cell-7 pre-incubated DARA only agglutination with TBS 2 Cell-7 pre-incubated DARA only no agglutination with DARA-F(ab).sub.2 3 Cell-11 pre-incubated DARA only agglutination with TBS 4 Cell-11 pre-incubated DARA only no agglutination with DARA-F(ab).sub.2 5 Cell-7 pre-incubated anti-s serum only agglutination with TBS 6 Cell-7 pre-incubated anti-s serum only agglutination with DARA-F(ab).sub.2 7 Cell-11 pre-incubated anti-s serum only no agglutination with TBS 8 Cell-11 pre-incubated anti-s serum only no agglutination with DARA-F(ab).sub.2 9 Cell-7 pre-incubated DARA + anti-s serum agglutination with TBS 10 Cell-7 pre-incubated DARA + anti-s serum agglutination with DARA-F(ab).sub.2 11 Cell-11 pre-incubated DARA + anti-s serum agglutination with TBS 12 Cell-11 pre-incubated DARA + anti-s serum no agglutination with DARA-F(ab).sub.2
(10) Wells 1 to 4 show comparative reactions containing no serum with anti-blood group antibody, but anti-CD38 antibody DARA and anti-human antibody (Coombs) only. These results show that the anti-CD38 antibody DARA leads to an agglutination of the untreated erythrocytes and that pre-treatment of erythrocytes by adding DARA-F(ab).sub.2 results in abolishment of the agglutination.
(11) Wells 5 to 8 show reactions containing only serum with anti-blood group antibody, but no anti-CD38 antibody DARA. These results show that the agglutination of s-positive Cell-7 by anti-s serum and the non-agglutination of s-negative Cell-11 with anti-s serum are not affected by the pre-incubation of the erythrocytes with the DARA-F(ab).sub.2 binding peptide.
(12) Well 9 shows that in the presence of the anti-s serum, without pre-treatment of erythrocytes by a binding peptide, an antibody (DARA) directed against a non-blood group antigen (CD38), or an anti-blood group antibody, currently anti-s, results in agglutination.
(13) Well 10 shows that adding a binding peptide that has no human Fc-region (DARA-F(ab).sub.2) and which is directed against a first antigen, exemplified by CD38, does not influence the binding of the anti-s serum antibody to the erythrocytes.
(14) Well 11 shows that in the presence of the anti-s serum, without pre-treatment of erythrocytes by a binding peptide, an antibody (DARA) directed against a non-blood group antigen (CD38) in a serum (anti-s) results in agglutination also for cells which do not bear the blood group antigen s.
(15) Well 12 shows that adding a binding peptide that has no human Fc-region (DARA-F(ab).sub.2) and which is directed against a first antigen, exemplified by CD38, does not influence the non-binding of the anti-s serum antibody to the erythrocytes which do not bear the s antigen.
(16) Accordingly, the addition of a binding peptide which does not have a human Fc-region and which is specific for a first antigen present on blood cell membranes results in the specific masking of the first antigen on the blood cell membranes without affecting the binding or non-binding, respectively, of other antibodies of a serum.
Example 2: Analysis of Anti-Blood Group Antibodies in Serum
(17) As described in Example 1, the therapeutic anti-CD38 antibody DARA was used as an antibody directed against the exemplary first antigen CD38, and DARA-F(ab).sub.2 was used as the binding peptide specific for the first antigen. The blood cell membranes were represented by erythrocytes Cell-2 (Kell positive, Fy(a) negative) and Cell-4 (Kell negative, Fy(a) positive) using human anti-Kell serum (obtained from Grifols, 1:2 dilution) or human anti-Fy(a) serum (obtained from Biolith, 1:2 dilution). Binding of serum antibody to the erythrocytes was detected by the agglutination reaction according to Example 1.
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(19) TABLE-US-00002 well No. cell serum reaction 1 Cell-2 pre-incubated anti-Kell serum only agglutination with TBS 2 Cell-2 pre-incubated anti-Kell serum only agglutination with DARA-F(ab).sub.2 3 Cell-4 pre-incubated anti-Kell serum only no agglutination with TBS 4 Cell-4 pre-incubated anti-Kell serum only no agglutination with DARA-F(ab).sub.2 5 Cell-2 pre-incubated DARA only agglutination with TBS 6 Cell-2 pre-incubated DARA only no agglutination with DARA-F(ab).sub.2 7 Cell-4 pre-incubated DARA only agglutination with TBS 8 Cell-4 pre-incubated DARA only no agglutination with DARA-F(ab).sub.2 9 Cell-2 pre-incubated anti-Fy(a) serum only no agglutination with TBS 10 Cell-2 pre-incubated anti-Fy(a) serum only no agglutination with DARA-F(ab).sub.2 11 Cell-4 pre-incubated anti-Fy(a) serum only agglutination with TBS 12 Cell-4 pre-incubated anti-Fy(a) serum only agglutination with DARA-F(ab).sub.2 13 Cell-2 pre-incubated DARA + anti-Kell serum agglutination with TBS 14 Cell-2 pre-incubated DARA + anti-Kell serum agglutination with DARA-F(ab).sub.2 15 Cell-4 pre-incubated DARA + anti-Kell serum agglutination with TBS 16 Cell-4 pre-incubated DARA + anti-Kell serum no agglutination with DARA-F(ab).sub.2 17 Cell-2 pre-incubated DARA + agglutination with TBS anti-Fy(a) serum 18 Cell-2 pre-incubated DARA + no agglutination with DARA-F(ab).sub.2 anti-Fy(a) serum 19 Cell-4 pre-incubated DARA + agglutination with TBS anti-Fy(a) serum 20 Cell-4 pre-incubated DARA + agglutination with DARA-F(ab).sub.2 anti-Fy(a) serum
(20) Wells 1 to 4 show that the anti-Kell serum agglutinates only the erythrocytes depending on the presence of the specific blood group antigen Kell, independent of the addition of the binding peptide DARA-F(ab).sub.2 prior to contacting the erythrocytes with the human serum.
(21) Wells 9 to 12 show that the anti-Fy(a) serum agglutinates only the erythrocytes depending on the presence of the specific blood group antigen Fy(a), independent of the addition of the binding peptide DARA-F(ab).sub.2 prior to contacting the erythrocytes with the human serum.
(22) This shows also for blood group antigens Kell and Fy(a) that the binding of the respective specific antibodies from serum is not influenced by the binding of the binding peptide to the blood cell membranes prior to their contact with the serum to be analysed.
(23) Wells 5 to 8 show that the antibody directed to a first antigen which is present on the blood cell membranes, results in binding of the antibody to the cells, and that this binding is prevented by addition of a binding peptide specific for the first antigen prior to contact with the antibody, and also that the cross-linking activity of anti-human antibody (Coombs) as used in the gel-card assay is not present with the binding peptide which has no human Fc-region. The further wells show that the cross-linking activity of anti-human antibody (Coombs) for anti-blood group antibodies of the serum that bind to the blood cell membranes is not influenced by the added binding peptide of the invention.
(24) Wells 13, 15, 17, and 19 show that the erythrocytes without pre-incubation with a binding peptide directed against a first antigen are bound by an antibody directed against the first antigen, resulting in agglutination, independent from the anti-blood group specificity of the serum contacted with the erythrocytes. Wells 14, 16, 18, and 20 show that the addition of the binding peptide that lacks a human Fc-region and has specificity for a first antigen prevents binding of an antibody present in the serum, which antibody is directed against the first antigen, and show that the binding of anti-blood group antibodies of the serum to the blood cell membranes is not affected by the prior addition of the binding peptide which is specific for the first antigen, which is not a blood group antigen recognized by the anti-blood group antibodies of the serum.
Example 3: Inhibition of DARA in Patient Serum
(25) Corresponding to Example 1, the therapeutic anti-CD38 antibody DARA was contained in the serum as an antibody directed against the exemplary first antigen CD38, which serum was obtained from a patient treated with the therapeutic anti-CD38 antibody DARA. DARA-F(ab).sub.2 was used as the binding peptide specific for the first antigen. The blood cell membranes were represented by erythrocytes Cell-9 and Cell-11. Binding of serum antibody to the erythrocytes was detected by the agglutination reaction according to Example 1.
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(27) TABLE-US-00003 Well No. Cell serum reaction 1 Cell-9 pre-incubated DARA only agglutination with TBS 2 Cell-9 pre-incubated DARA only no agglutination with DARA-F(ab).sub.2 3 Cell-11 pre-incubated DARA only agglutination with TBS 4 Cell-11 pre-incubated DARA only no agglutination with DARA-F(ab).sub.2 5 Cell-9 pre-incubated Patient serum agglutination with TBS 6 Cell-9 pre-incubated Patient serum no agglutination with DARA-F(ab).sub.2 7 Cell-11 pre-incubated Patient serum agglutination with TBS 8 Cell-11 pre-incubated Patient serum no agglutination with DARA-F(ab).sub.2
(28) Cell-9 carries the typed antigens Rhc, Rhe, Cellano, Kp(b), Kp(a), Fy(a), Fy(b), Jk(b), Le(b), P1, M, S, s, and Lu(b). Cell-11 carries the typed antigens Rhc, Rhe, Cellano, Kp(b), Fy(b), Jk(a), Le(b), P1, N, S, Lu(b), Xg(a), Bga(w)
(29) Wells 1 to 4 show comparative reactions containing no serum with anti-blood group antibody, but anti-CD38 antibody DARA and anti-human antibody (Coombs) only. These results show that the anti-CD38 antibody DARA leads to an agglutination of all the untreated erythrocytes and that pre-treatment of erythrocytes by adding DARA-F(ab).sub.2 results in abolishment of this agglutination.
(30) Wells 5 to 8 show comparative reactions containing patient serum with unknown or no anti-blood group antibodies, but anti-CD38 antibody due to treatment with DARA. These results show that the agglutination of the untreated erythrocytes due to the anti-CD38 antibody DARA in a patient serum can be abolished by pre-treatment of erythrocytes by adding DARA-F(ab).sub.2. The reactions of wells 5 to 8 show that this patient is negative for antibodies against the antigens on Cell-9 and Cell-11.
(31) The results demonstrate that pre-treating the blood cell membranes, represented by erythrocytes, with a binding peptide specific for the same first antigen (CD38) of the blood cell membranes, which binding peptide contains no human Fc-region, rendered the serum antibody inactive that is specific for the first antigen. For the determination of blood group antibodies, the pre-incubation of the blood cell membranes with such a binding peptide turned a non-conclusive result into a conclusive result, as the all-positive agglutination reactions were turned into reactions specifically showing the effect of other serum antibodies.