WO1983003477A1 - Blood cell grouping reagent - Google Patents
Blood cell grouping reagent Download PDFInfo
- Publication number
- WO1983003477A1 WO1983003477A1 PCT/GB1983/000081 GB8300081W WO8303477A1 WO 1983003477 A1 WO1983003477 A1 WO 1983003477A1 GB 8300081 W GB8300081 W GB 8300081W WO 8303477 A1 WO8303477 A1 WO 8303477A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- monoclonal
- cells
- monoclonal antibodies
- serum
- blood
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/34—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
Definitions
- the present invention relates to ABO blood cell grouping reagents.
- ABO incompatibility is a potentially lethal barrier in transfusion therapy and in tissue transplantation. All donor blood and potential recipients for transfusion or tissue transplantation are therefore routinely ABO grouped as this is the only way to ensure maximum safety for these patients.
- ABO blood cell grouping is conventionally performed by testing the red cells of the blood to be typed for the presence or absence of antibodies and antigens. The presence of the latter is generally determined by adding an anti-serum (a blood serum containing known antibodies to the A and B red cell antigens) to the red cells in saline suspension and looking for agglutination.
- the anti-sera employed in the grouping of ABO blood contain either anti- A anti- bodies (Group B serum) or anti-B antibodies (Group A serum) or both anti-A and anti-B antibodies (Group 0 serum). Of these, Group 0 serum is used to check that the anti-A and anti-B tests, that are used in all routine ABO groupings of blood donations and patients, are correct. The main factors affecting the choice of ABO reagents are,
- an ABO blood cell grouping reagent comprising, in admixture, one or more anti-A monoclonal antibodies and one or more anti-B monoclonal antibodies.
- the present ABO blood cell grouping reagent is an anti-A/B grouping reagent which detects A antigenic determinants and B antigenic determinants on red blood cells.
- an anti-A monoclonal antibody incorporates an anti-A,B monoclonal antibody, provided the anti-A,B monoclonal antibody is potent enough to cause a macroscopic reaction (by spin-tube, tube sedimentation, or tile/slide methods) with red blood cells possessing one or more A-type antigenic determinants.
- an anti-B monoclonal antibody incorporates an anti-A,B monoclonal antibody, provided the anti-A,B monoclonal, antibody is potent enough to cause a macroscopic reaction (byspin-tube, tube sedimentation or tile/slide methods) with red blood cells possessing one or more B-type antigenic determinants.
- a 'sufficient agglutinating quality' will be an agglutinating quality as good as or better than that displayed by current UK National Health Service group 0 reagents.
- 'Routine blood typing methods' are set out in full in "Techniques in blood grouping" by I Dunsford and C C Bowley, 2nd Edn., 1967.
- the reagent may also incorporate one or more monoclonal antibodies (in particular monoclonal anti-A,B antibodies) that are specific for the antigenic determinants on Ax-type red blood cells (Ax activity) and Bx-type red blood cells (Bx activity) (or other weak B variants).
- a reagent that possesses Ax activity is particularly useful since such a reagent should prevent the mistyping of certain weak A-type blood cells as group 0.
- the monoclonal antibodies are derived from hybrid myelomas produced by the fusion of mouse myeloma, cells, especially NSI (abbreviated from P3-NSI/1-Ag4-1, G Kohler, S C Howe and C Milstein, Eur J Immunol,1976, 6, 292) or NSO/1(G.Galfre and C Milstein, Methods in Enzymology, 1981,73,3), both available on request from the MRC Laboratory of Molecular Biology, Hills Road, Cambridge), with spleen or other immunocyte cells from mice immunissd with human antigen A,human antigen B, or human antigen A,B.
- NSI abbreviated from P3-NSI/1-Ag4-1, G Kohler, S C Howe and C Milstein, Eur J Immunol,1976, 6, 292
- NB1/19.112.28 are mixed.
- a monoclonal anti-A (3D3), derived from the hybrid myeloma A15-3D3. 92. 00, and monoclonal anti-B (NB1/19) are mixed.
- the monoclonal antibodies used to form the present reagent may be separated either from the ascitic fluids of tumours produced in animals, generally mice, or from tissue culture supernatants.
- the present reagent is not an anti-serum as previously understood since the antibodies are not contained in a serum.
- the medium surrounding the monoclonal antibodies will still, in many cases, contain a certain proportion of serum.
- the serum will often be present in the ascitic fluid as collected, whilst in the latter case the serum will be added to the tissue culture to provide nutrients for cell growth.
- the present reagent may also contain a certain proportion of conventional Group 0 serum. The addition of this serum is to provide the present reagent with A x activity, especially in cases where the mixture of monoclonal antibodies alone lack this activity. In a particularly preferred embodiment a one-third volume of Group 0 serum is added.
- the present reagent will be used primarily to check that anti-A and anti-B tests (for antigens A and B) are correct.
- a process for the grouping of ABO blood cells comprising adding to a suspension of cells in sodium chloride solution, a blood cell grouping reagent which comprises, in admixture, one or more anti-A monoclonal antibodies and one or more anti-B monoclonal antibodies.
- anti-A and anti-B monoclonal antibodies incorporate certain anti-A,B monoclonal antibodies, as defined above.
- the preferred monoclonal antibodies for use in this process are also listed above.
- the reagent may also contain a certain proportion of conventional group 0 serum, especially if none of the monoclonal antibodies employed exhibit Ax activity.
- the ABO blood cell blended grouping reagents of the present invention are as stable as the individual antibodies used in the blend when stored at 4oC or lower temperatures.
- the cell grouping test is generally continued by observing whether or not agglutination of the cells takes place.
- the present reagent contains an antibody specific for one or more of the antigens of the cells under test, then agglutination will take place.
- Such agglutination is a visible reaction and therefore may be followed visually in a qualitative or quantitative manner.
- the present reagent may be provided for use in the above process in the form of a blood cell grouping kit.
- the anti-A monoclonal antibodies and the anti-B monoclonal antibodies are provided in the form of a mixture.
- the anti-A monoclonal antibodies and the anti-B monoclonal antibodies are provided in separate containers together with instructions on the method of mixing.
- either of the previous kits also contains group 0 serum, either in admixture with or separate from the monoclonal antibodies.
- the myeloma NS1 produces K light chains which, although not secreted by the myeloma, can be 'mixed' with the light chains of spleen cell origin and the antibody secreted as mixed chain types called HLK.
- Full antibody activity requires both light and heavy chains, hence HLK antibody molecules are defective as some of the light chains are non-antibody myeloma K chains and thus reduce the antibody binding efficiency of the molecules. Therefore it is obviously desirable to select HL variants to obtain the best agglutinating antibodies. Recenctly myelomas which do not produce K chains, e.g. NSO/1, have become available and this eliminates the HLK problem.
- Comoosition is stablished by radioautograph of electrophoretic runs detecting the C 14 lysine incorporated into the antibodies secreted by the hybrid-myeloma cells.
- SDS-PAGE The separation is based on molecular sieving of the different sized components that have a similar negative charge by being coated with SDS.
- Antibody dilutions were made in saline and titration results scored. Elution was by the heat method, and enhancement tests used 2% pre-papainised cell suspensions or 20% bovine albumin by the displacement method.
- C3H/He-mg mice were immunised with tissue culture cells (HT-29 from Dr J Fogh, Sloan Kettering Institute) from a human group
- Spleen cells (10 8 ) from the immunised mice were fused with mouse myeloma cells (10 7 ) by use of the polyethylene glycol method (Weir, Handbook of experimental immunology, 3rd Edn, vol II).
- the myeloma cells were non-secretor P3-NS1/1-Ag4-1 selected enzyme deficient cells that die in HAT medium.
- the culture supematants were tested 2 weeks post fusion and those cells with antibody binding activity on HT-29 cells were cloned on soft agar and grown to larger volumes, recloned, regrown and divided into aliquots for larger scale antibody production and liquid nitrogen storage of the selected cell lines.
- the established anti-A secreting clone (6D4) was grown in Dulbecco's modified Eagle's medium (BMEH, Gibco Biocult) supplemented with feotal calf serum (FCS, Sera-Lab). The concentration of FCS is gradually reduced from 15 to 5%.
- the supplemented tissue culture medium was free of anti-A or anti-B activity and A and B substances (antigens).
- the tissue culture supernatants were centrifuged to remove cells and debris, supplemented with 10mM Hepes and 0.1% sodium azide and stored at 4o, -20o or -40oC. Aliquots were concentrated 5 fold,
- mice Male BIO BR mice (OLAC) were immunised with A 1 cells followed by several injections of human blood group A substance. Spleen cells (10 8 ) from the immunised mice were fused with mouse myeloma cells (10 7 ) by the use of the polyethylene glycol method. The myeloma cells were non-secretor NSO/1 derived from P3-NS1/1/Ag4/1 selected enzyme deficient cells that die in HAT medium.
- the culture supernatants were tested, beginning 2 week post fusion and those cells producing anti-A were selected by agglutina tion tests, then cloned on soft agar, grown to larger volumes, recloned, regrown and divided into aliquots for larger scale antibody production and liquid nitrogen storage of the selected cell lines.
- the established anti-A secreting clone (3D3) was grown in Dulbecco's modified Eagle's medium (DMEM, Gibco Biocult), supplemented with feotal calf serum (FCS, Serolab). The concentration of FCS was gradually reduced from 15% to 2.5%. The supplemented tissue culture medium was free of anti-A or anti-B activity and A or B substances.
- DMEM Dulbecco's modified Eagle's medium
- FCS feotal calf serum
- mice serum samples were assayed for anti-B activity after absorption with group 0 cells to remove anti-species antibodies.
- a mouse with an anti-B agglutination titre of 1:8 after absorption was chosen and injected intraperitoneally with 100 ⁇ g group B substance (donated by Dr W Watkins, MRC Clinical Research Centre, Harrow) in 0.1ml complete Freund's adjuvant (Difco Bacto). This injection was repeated after 5 weeks and boosted after a further 9 weeks with 200 ⁇ g B substance in 0.1ml saline injected intravenously. 3 days later, the spleen was removed and a cell suspension was prepared.
- Spleen cells (10 8 ) were fused with mouse myeloma NS1 cells (10 7 ) using polyethylene glycol.
- Growing cell hybrids were selected by their ability to produce specific anti-B activity, detected in the culture supernatant by agglutination assays (I Dunsford and C C Bowley, Techniques in blood grouping, 2nd Edn, 1967) using human A, B and O erythrocytes.
- Anti-B secreting hybrids were cloned twice on soft agar, and grown up into 1 litre spinner cultures in
- DMEM Dulbecco's Modified Eagle's Medium
- FCS feotal calf serum
- Tissue culture supernatant containing the monoclonal antibody was prepared by centrifugation to remove cells and debris, filtration through Millipore filters and addition of 10mM Hepes buffer and 0.1% sodium azide. Aliquots of each batch were then stored at 4°C for routine use or -20°C for stocks.
- Example 4 Preparation of monoclonal anti-A,B (A15/1A4.7) Male B10 BR mice (OLAC) were immunised with A 1 cells followed by several injections of human blood group A substance. Spleen cells (10 8 ) from the immunised mice were fused with mouse myeloma cells(10 7 ) by the use of the polyethylene glycol method. The myeloma cells were non-secretor NSO/1 derived from P3-NS1/1/Ag4/1 selected enzyme deficient cells that die in HAT medium.
- the culture supernatants were tested, beginning 2 week post fusion and those cells producing anti- A,B were selected by agglutination tests, then cloned on soft agar, grown to larger volumes, recloned, regrown and divided into aliquots for larger scale antibody production and liquid nitrogen storage of the selected celllines.
- FCS feotal calf serum
- the supplemented tissue culture medium was free of anti-A or anti-B activity and A or B substances.
- Example 5 Blending monoclonal anti-A and anti-B reagents.
- An ABO blood cell grouping reagent was prepared by mixing (in a 1:1:1 (v/v/v)ratio) monoclonal anti-A supernatant (5 fold concentrate, Example 1), monoclonal anti-B supernatant(Example 3) and group 0 serum (from a Hyperimmunised group 0 donor). Titration scores and avidity times for this mixture were compared with those of a group 0 serum obtained from a hyperimmunised group 0 donor (and in the latter case with commercial and NHS group 0 sera). Results are given in Tables 1 and 2.
- An AB0 blood cell grouping reagent was prepared by mixing (in a 1 : 1 (v/v) ratio) monoclonal anti-A supernatant (2 fold concentrate, Example 2) and monoclonal anti-B supernatant (2 fold concentrate, Example 3) . Titration scores for this mixture are given in Table 3. The avidity scores for the mixture, which were compared to commercial sera, are given in Table 4.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Un réactif de détermination des groupes sanguins AB0 comprend un mélange d'un ou plusieurs anticorps monoclonaux anti-A et d'un ou plusieurs anticorps monoclonaux anti-B. Un anticorps monoclonal anti-A incorpore un anticorps monoclonal anti-A, B à condition que l'anticorps anti-A,B soit assez puissant pour provoquer une réaction macroscopique avec des cellules sanguines ayant un ou plusieurs déterminants antigènes de type A. De même, un anticorps monoclonal anti-B incorpore un anticorps monoclonal anti-A,B B, anti-A à condition que l'anticorps anti-A,B soit assez puissant pour provoquer une réaction macroscopique avec des cellules sanguines ayant un ou plusieurs antigènes de type B. Il est possible d'utiliser tout mélange d'anti-corps monoclonaux anti-A et anti-B ayant une qualité d'agglutination suffisante pour donner des résultats sûrs (par la présence d'agglutinogènes A et B) selon les procédés habituels de détermination du groupe sanguin. De préférence, les anticorps monoclonaux sont dérivés de myélomes hybrides produits par la fusion d'une lignée cellulaire mère, spécialement NSI (abréviation de P3-NS1/1-Ag 4-1) ou NSO/1, avec des cellules de la rate ou autres immunocytes de souris immunisées avec de l'agglutinogène humain A ou B ou A,B. Dans un mode préférentiel de réalisation, un anti-A monoclonal (6D4 ou 3D3) et un anti-B monoclonal (NB1/19), dérivés respectivement du myélome hybride 6D4.12.3, A15 3D3.92.00 et NB1/19.112.28, sont mélangés. Le réactif peut également contenir une certaine proportion de sérum conventionnel du groupe 0. L'addition de ce sérum vise à donner au réactif une activité Ax, spécialement si cette activité fait défaut dans le mélange monoclonal. Dans un mode préférentiel de réalisation, on ajoute un tiers de volume de sérum du groupe 0.An AB0 blood grouping reagent comprises a mixture of one or more anti-A monoclonal antibodies and one or more anti-B monoclonal antibodies. An anti-A monoclonal antibody incorporates an anti-A,B monoclonal antibody provided that the anti-A,B antibody is potent enough to cause a macroscopic reaction with blood cells having one or more type A antigenic determinants. , an anti-B monoclonal antibody incorporates an anti-A,B B, anti-A monoclonal antibody provided that the anti-A,B antibody is potent enough to cause a macroscopic reaction with blood cells having one or more type antigens B. It is possible to use any mixture of anti-A and anti-B monoclonal antibodies having sufficient agglutination quality to give reliable results (by the presence of agglutinogens A and B) according to the usual methods blood group determination. Preferably, the monoclonal antibodies are derived from hybrid myelomas produced by the fusion of a parent cell line, especially NSI (abbreviation of P3-NS1/1-Ag 4-1) or NSO/1, with cells of the spleen or other immunocytes from mice immunized with human agglutinogen A or B or A,B. In a preferred embodiment, a monoclonal anti-A (6D4 or 3D3) and a monoclonal anti-B (NB1/19), respectively derived from hybrid myeloma 6D4.12.3, A15 3D3.92.00 and NB1/19.112.28, are mixed. The reagent may also contain a certain proportion of conventional group 0 serum. The addition of this serum is intended to give the reagent an Ax activity, especially if this activity is lacking in the monoclonal mixture. In a preferred embodiment, a third volume of group 0 serum is added.
Description
BLOOD CELL GROUPING REAGENT
The present invention relates to ABO blood cell grouping reagents.
ABO incompatibility is a potentially lethal barrier in transfusion therapy and in tissue transplantation. All donor blood and potential recipients for transfusion or tissue transplantation are therefore routinely ABO grouped as this is the only way to ensure maximum safety for these patients.
ABO blood cell grouping is conventionally performed by testing the red cells of the blood to be typed for the presence or absence of antibodies and antigens. The presence of the latter is generally determined by adding an anti-serum (a blood serum containing known antibodies to the A and B red cell antigens) to the red cells in saline suspension and looking for agglutination. The anti-sera employed in the grouping of ABO blood contain either anti- A anti- bodies (Group B serum) or anti-B antibodies (Group A serum) or both anti-A and anti-B antibodies (Group 0 serum). Of these, Group 0 serum is used to check that the anti-A and anti-B tests, that are used in all routine ABO groupings of blood donations and patients, are correct. The main factors affecting the choice of ABO reagents are,
(a) The specificity of the reagent for the appropriate antigen.
(b) The stability of the reagent under the conditions of use.
(c) The potency or agglutinating quality which must be sufficient to give reliable results by the routine methods employed.
(d) The guaranteed availability of sufficient volumes to meet the commitments of the transfusion service, and
(e) The cost of production which must be as low as nossible.
At present the National Health Service obtains anti-sera either by screening the sera of donor blood samples or by hyperimmunisation of volunteer donors. By contrast commercial sources and most European and North American countries obtain sera only from hyperimmunised donors. In both cases the supply of potent group 0 serum is limited, and in the latter case expensive. This limited availability cannot be remedied by blending the more readily obtainable group A and group B sera, since this leads to the loss of some anti-A and anti-B activity by cross neutralisation. It is the aim of the present invention to provide a new ABO blood cell grouping reagent that may be used in place of conventional group 0 serum and that is both readily available and cheap to produce.
According to the present invention there is provided an ABO blood cell grouping reagent comprising, in admixture, one or more anti-A monoclonal antibodies and one or more anti-B monoclonal antibodies.
The present ABO blood cell grouping reagent is an anti-A/B grouping reagent which detects A antigenic determinants and B antigenic determinants on red blood cells.
In the present specification an anti-A monoclonal antibody incorporates an anti-A,B monoclonal antibody, provided the anti-A,B monoclonal antibody is potent enough to cause a macroscopic reaction (by spin-tube, tube sedimentation, or tile/slide methods) with red blood cells possessing one or more A-type antigenic determinants. Moreover, an anti-B monoclonal antibody incorporates an anti-A,B monoclonal antibody, provided the anti-A,B monoclonal, antibody is potent enough to cause a macroscopic reaction (byspin-tube, tube sedimentation or tile/slide methods) with red blood cells possessing one or more B-type antigenic determinants.
Any mixture of monoclonal anti-A and anti-B antibodies that has sufficient agglutinating quality to give reliable results (for the presence of antigens A and B) by the routine blood. typing methods may be used as an ABO reagent according to this invention. A 'sufficient agglutinating quality' will be an agglutinating quality as good as or better than that displayed by current UK National Health Service group 0 reagents. 'Routine blood typing methods' are set out
in full in "Techniques in blood grouping" by I Dunsford and C C Bowley, 2nd Edn., 1967.
In a further aspect of the present invention the reagent may also incorporate one or more monoclonal antibodies (in particular monoclonal anti-A,B antibodies) that are specific for the antigenic determinants on Ax-type red blood cells (Ax activity) and Bx-type red blood cells (Bx activity) (or other weak B variants). A reagent that possesses Ax activity is particularly useful since such a reagent should prevent the mistyping of certain weak A-type blood cells as group 0.
Preferably the monoclonal antibodies are derived from hybrid myelomas produced by the fusion of mouse myeloma, cells, especially NSI (abbreviated from P3-NSI/1-Ag4-1, G Kohler, S C Howe and C Milstein, Eur J Immunol,1976, 6, 292) or NSO/1(G.Galfre and C Milstein, Methods in Enzymology, 1981,73,3), both available on request from the MRC Laboratory of Molecular Biology, Hills Road, Cambridge), with spleen or other immunocyte cells from mice immunissd with human antigen A,human antigen B, or human antigen A,B.In onepart icularly preferred embodiment a monoclonal anti-A(6D4) and a monoclonal anti-B (NB1/19), derived respectfully from the hybrid myeloma 6D4. 12.3 and NB1/19.112.28 are mixed. In another preferred embodiment a monoclonal anti-A (3D3), derived from the hybrid myeloma A15-3D3. 92. 00, and monoclonal anti-B (NB1/19) are mixed.
The monoclonal antibodies used to form the present reagent may be separated either from the ascitic fluids of tumours produced in animals, generally mice, or from tissue culture supernatants.
It will be seen from the above that the present reagent is not an anti-serum as previously understood since the antibodies are not contained in a serum. However the medium surrounding the monoclonal antibodies will still, in many cases, contain a certain proportion of serum. In the former case the serum will often be present in the ascitic fluid as collected, whilst in the latter case the serum will be added to the tissue culture to provide nutrients for cell growth. In addition to this serum the present reagent may also contain a certain proportion of conventional Group 0 serum. The addition of this serum is to provide the present reagent with Ax activity, especially in cases where the mixture of monoclonal antibodies alone lack this activity.
In a particularly preferred embodiment a one-third volume of Group 0 serum is added.
The present reagent will be used primarily to check that anti-A and anti-B tests (for antigens A and B) are correct. In order to facilitate this test there is also provided a process for the grouping of ABO blood cells comprising adding to a suspension of cells in sodium chloride solution, a blood cell grouping reagent which comprises, in admixture, one or more anti-A monoclonal antibodies and one or more anti-B monoclonal antibodies. Once again the terms anti-A and anti-B monoclonal antibodies incorporate certain anti-A,B monoclonal antibodies, as defined above. Moreover the preferred monoclonal antibodies for use in this process are also listed above. Further, the reagent may also contain a certain proportion of conventional group 0 serum, especially if none of the monoclonal antibodies employed exhibit Ax activity.
The ABO blood cell blended grouping reagents of the present invention are as stable as the individual antibodies used in the blend when stored at 4ºC or lower temperatures. Once the present blood cell grouping reagent has been added to the blood cell suspension in sodium chloride solution, the cell grouping test is generally continued by observing whether or not agglutination of the cells takes place. Generally, if the present reagent contains an antibody specific for one or more of the antigens of the cells under test, then agglutination will take place. Such agglutination is a visible reaction and therefore may be followed visually in a qualitative or quantitative manner. On the other hand, if the present reagent does not contain an antibody specific for the blood cells antigens then generally,unless a cross reaction takes place,no agglutination will occur.(Cross reaction refers to both false reactions and auto antibody sensitised cells). For convenience, the present reagent may be provided for use in the above process in the form of a blood cell grouping kit. In one embodiment of this kit the anti-A monoclonal antibodies and the anti-B monoclonal antibodies are provided in the form of a mixture. In a second embodiment the anti-A monoclonal antibodies and the anti-B monoclonal antibodies are provided in separate containers together with instructions on the method of mixing. In a third embodiment either of the previous kits also contains group 0 serum, either in admixture with or separate from the monoclonal antibodies.
The reagent, process and kit of the present invention will now be described by way of example only,
Materials and Methods
Analysis to determine antibody heavy and light chains The myeloma NS1 produces K light chains which, although not secreted by the myeloma, can be 'mixed' with the light chains of spleen cell origin and the antibody secreted as mixed chain types called HLK. Full antibody activity requires both light and heavy chains, hence HLK antibody molecules are defective as some of the light chains are non-antibody myeloma K chains and thus reduce the antibody binding efficiency of the molecules. Therefore it is obviously desirable to select HL variants to obtain the best agglutinating antibodies. Recenctly myelomas which do not produce K chains, e.g. NSO/1, have become available and this eliminates the HLK problem. Comoosition is stablished by radioautograph of electrophoretic runs detecting the C14 lysine incorporated into the antibodies secreted by the hybrid-myeloma cells. There are two types of procedure for separating the antibody compartments in poly-acrylamide gel slabs for subsequent radioautograph analysis: (i) Iso-electric focussing (TEF). The separation is based on pH gradient separating components having different iso-electric points with prior reduction by 2-mercapto ethanol to break the interchain links.
(ii) Electrophoresis of the reduced antibodies with sodium dodecyl sulphate polyacrylamide slab gel electrophoresis
(SDS-PAGE). The separation is based on molecular sieving of the different sized components that have a similar negative charge by being coated with SDS.
Serum reagents NHS anti-sera prepared by the Blood Group Reference Laboratory
(BGRL) from sera referred by regional transfusion centres.
Commercial antisera from several sources.
Haemagglutination tests
These were tube tests as set out in Dunsford and Bowley. Red cells were from ACD or clotted samples received in the transfusion centre. Cells were washed X4 in isotonic saline for comparative titration studies, but otherwise used by preparing a 2-3% suspension in saline. Cells were used at 20% for slide tests and 5% for spin tube tests.
Antibody dilutions were made in saline and titration results scored. Elution was by the heat method, and enhancement tests used 2% pre-papainised cell suspensions or 20% bovine albumin by the displacement method.
Example 1. Preparation of monoclonal anti-A (6D4)
C3H/He-mg mice (OLAC) were immunised with tissue culture cells (HT-29 from Dr J Fogh, Sloan Kettering Institute) from a human group
A colon carcinoma case. Spleen cells (108) from the immunised mice were fused with mouse myeloma cells (107) by use of the polyethylene glycol method (Weir, Handbook of experimental immunology, 3rd Edn, vol II). The myeloma cells were non-secretor P3-NS1/1-Ag4-1 selected enzyme deficient cells that die in HAT medium.
The culture supematants were tested 2 weeks post fusion and those cells with antibody binding activity on HT-29 cells were cloned on soft agar and grown to larger volumes, recloned, regrown and divided into aliquots for larger scale antibody production and liquid nitrogen storage of the selected cell lines.
The established anti-A secreting clone (6D4) was grown in Dulbecco's modified Eagle's medium (BMEH, Gibco Biocult) supplemented with feotal calf serum (FCS, Sera-Lab). The concentration of FCS is gradually reduced from 15 to 5%. The supplemented tissue culture medium was free of anti-A or anti-B activity and A and B substances (antigens).
The tissue culture supernatants were centrifuged to remove cells and debris, supplemented with 10mM Hepes and 0.1% sodium azide and stored at 4º, -20º or -40ºC. Aliquots were concentrated 5 fold,
8 fold and 18 fold by ultra-filtration on an Amicon filter (PM 10). and stored at 4° and -20ºC.
Example 2. Preparation of monoclonal anti-A (3D3)
Male BIO BR mice (OLAC) were immunised with A1 cells followed by several injections of human blood group A substance. Spleen cells (108) from the immunised mice were fused with mouse myeloma cells (107) by the use of the polyethylene glycol method. The myeloma cells were non-secretor NSO/1 derived from P3-NS1/1/Ag4/1 selected enzyme deficient cells that die in HAT medium.
The culture supernatants were tested, beginning 2 week post fusion and those cells producing anti-A were selected by agglutina tion tests, then cloned on soft agar, grown to larger volumes, recloned, regrown and divided into aliquots for larger scale antibody production and liquid nitrogen storage of the selected cell lines.
To prepare culture supernatants for testing, the established anti-A secreting clone (3D3) was grown in Dulbecco's modified Eagle's medium (DMEM, Gibco Biocult), supplemented with feotal calf serum (FCS, Serolab). The concentration of FCS was gradually reduced from 15% to 2.5%. The supplemented tissue culture medium was free of anti-A or anti-B activity and A or B substances.
Example 3. Preparation of monoclonal anti-B (NB1-19).
To find suitable mice, serum samples were assayed for anti-B activity after absorption with group 0 cells to remove anti-species antibodies. A mouse with an anti-B agglutination titre of 1:8 after absorption was chosen and injected intraperitoneally with 100 μg group B substance (donated by Dr W Watkins, MRC Clinical Research Centre, Harrow) in 0.1ml complete Freund's adjuvant (Difco Bacto). This injection was repeated after 5 weeks and boosted after a further 9 weeks with 200μg B substance in 0.1ml saline injected intravenously. 3 days later, the spleen was removed and a cell suspension was prepared.
Spleen cells (108) were fused with mouse myeloma NS1 cells (107) using polyethylene glycol. Growing cell hybrids were selected by their ability to produce specific anti-B activity, detected in the culture supernatant by agglutination assays (I Dunsford and C C Bowley, Techniques in blood grouping, 2nd Edn, 1967) using human A, B and O erythrocytes. Anti-B secreting hybrids were cloned twice on soft agar, and grown up into 1 litre spinner cultures in
Dulbecco's Modified Eagle's Medium (DMEM, Gibco Biocult) supplemented with 5% (v/v) feotal calf serum (FCS, Sera-Lab). The cloned hybrids were stored in liquid nitrogen.
Tissue culture supernatant containing the monoclonal antibody was prepared by centrifugation to remove cells and debris, filtration through Millipore filters and addition of 10mM Hepes buffer and 0.1% sodium azide. Aliquots of each batch were then stored at 4°C for routine use or -20°C for stocks.
Example 4. Preparation of monoclonal anti-A,B (A15/1A4.7) Male B10 BR mice (OLAC) were immunised with A1 cells followed by several injections of human blood group A substance. Spleen cells (108) from the immunised mice were fused with mouse myeloma cells(107) by the use of the polyethylene glycol method. The myeloma cells were non-secretor NSO/1 derived from P3-NS1/1/Ag4/1 selected enzyme deficient cells that die in HAT medium.
The culture supernatants were tested, beginning 2 week post fusion and those cells producing anti- A,B were selected by agglutination tests, then cloned on soft agar, grown to larger volumes, recloned, regrown and divided into aliquots for larger scale antibody production and liquid nitrogen storage of the selected celllines.
To prepare culture supernatants for testing, the established anti-A,B secreting clone (A15/1A4.7) was grown in Dulbecco's modified Eagle's medium (DMEM, Gibco Biocult), supplemented with feotal calf serum (FCS, Serolab). The concentration of FCS was gradually reduced from 15% to 2.5%.
The supplemented tissue culture medium was free of anti-A or anti-B activity and A or B substances.
Example 5. Blending monoclonal anti-A and anti-B reagents.
An ABO blood cell grouping reagent was prepared by mixing (in a 1:1:1 (v/v/v)ratio) monoclonal anti-A supernatant (5 fold concentrate, Example 1), monoclonal anti-B supernatant(Example 3) and group 0 serum (from a Hyperimmunised group 0 donor). Titration scores and avidity times for this mixture were compared with those of a group 0 serum obtained from a hyperimmunised group 0 donor (and in the latter case with commercial and NHS group 0 sera). Results are given in Tables 1 and 2.
Example 6. Blending monoclonal anti-A and anti-B reagents
An AB0 blood cell grouping reagent was prepared by mixing (in a 1 : 1 (v/v) ratio) monoclonal anti-A supernatant (2 fold concentrate, Example 2) and monoclonal anti-B supernatant (2 fold concentrate, Example 3) . Titration scores for this mixture are given in Table 3. The avidity scores for the mixture, which were compared to commercial sera, are given in Table 4.
Claims
1. An ABO blood grouping reagent comprising, in admixture, one or more anti-A monoclonal antibodies and one or more anti-B monoclonal antibodies.
2. An ABO blood grouping reagent according to claim 1 wherein one or more of the monoclonal antibodies has Ax activity.
3. An ABO blood grouping reagent according to claim 1 wherein one or more of the monoclonal antibodies has Bx activity.
4. An ABO blood grouping reagent according to claim 1 wherein the one or more monoclonal antibodies are derived from hybrid myelomas prepared by fusion of spleen cells with mouse myeloma cells selected from NSI cells and NSO cells.
5. An ABO blood grouping reagent according to claim 1 further comprising Group 0 serum.
6. A process for the grouping of ABO blood cells comprising adding to a suspension of cells in sodium chloride solution a blood cell grouping reagent according to claim 1 and detecting the agglutination or non-agglutination of the cells within the solution.
7. A blood cell grouping kit for use in the process of claim 6 comprising an ABO blood grouping reagent according to claim 1.
8. A blood cell grouping kit for use in the process of claim 6 comprising, in separate containers,
(a) one or more anti-A monoclonal antibodies, and
(b) one or nore anti-B monoclonal antibodies.
9. A blood cell grouping kit according to claim 8 further comprising Group 0 serum.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT83901088T ATE36606T1 (en) | 1982-03-29 | 1983-03-18 | BLOOD TYPE DETERMINATION REAGENTS. |
| JP58501140A JPS59500633A (en) | 1982-03-29 | 1983-03-18 | Classification reagent for blood cells |
| DE8383901088T DE3377745D1 (en) | 1982-03-29 | 1983-03-18 | Blood cell grouping reagent |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8209235 | 1982-03-29 | ||
| GB8209235 | 1982-03-29 | ||
| GB8305364830225 | 1983-02-25 | ||
| GB08305364A GB2117789B (en) | 1982-03-29 | 1983-02-25 | Abo blood grouping reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1983003477A1 true WO1983003477A1 (en) | 1983-10-13 |
Family
ID=26282419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/GB1983/000081 Ceased WO1983003477A1 (en) | 1982-03-29 | 1983-03-18 | Blood cell grouping reagent |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0103620B1 (en) |
| JP (1) | JPS59500633A (en) |
| DE (1) | DE3377745D1 (en) |
| GB (1) | GB2117789B (en) |
| WO (1) | WO1983003477A1 (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0138377A1 (en) * | 1983-09-16 | 1985-04-24 | The Wistar Institute | Lewis blood group phenotype assay |
| EP0223978A1 (en) * | 1985-10-11 | 1987-06-03 | Abbott Laboratories | Diagnostic test |
| US4764465A (en) * | 1984-04-26 | 1988-08-16 | Cetus Corporation | Human monoclonal antibody against group A red blood cells |
| EP0115476A3 (en) * | 1983-01-21 | 1988-10-12 | Monocarb Ab | Monoclonal antibody formulation for diagnostic use |
| EP0232706A3 (en) * | 1986-01-10 | 1988-11-30 | Sloan-Kettering Institute For Cancer Research | Blood group antigen panel |
| US5583003A (en) * | 1989-09-25 | 1996-12-10 | Agen Limited | Agglutination assay |
| RU2138812C1 (en) * | 1998-11-11 | 1999-09-27 | Сахаров Роман Сергеевич | METHOD OF PREPARING SPECIFICALLY REACTING IN REACTION ABSORPTION-ELUTION SERA ANTI-A (α), ANTI-B (β) IN DETECTION OF ANTIGENS A, B IN HUMAN EXCRETION TRACES |
| CN102944684A (en) * | 2012-11-07 | 2013-02-27 | 武汉康莱博生物科技有限公司 | A and B-type blood type resistant sizing reagent |
| WO2020106623A1 (en) | 2018-11-19 | 2020-05-28 | New Health Sciences, Inc. | Methods for the preservation of reagent red blood cells using carbon monoxide |
| WO2021046424A2 (en) | 2019-09-05 | 2021-03-11 | Hemanext Inc. | Methods for the preservation of reagent red blood cells using carbon monoxide |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2582103B1 (en) * | 1985-03-19 | 1989-05-05 | Canavaggio Michel | METHOD FOR DETERMINING A BIOLOGICAL SUBSTANCE IN A SAMPLE |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1981002104A1 (en) * | 1980-01-28 | 1981-08-06 | S Svensson | Isolation of carbohydrates from cell tissue |
| WO1982003089A1 (en) * | 1981-03-06 | 1982-09-16 | Lennox Edwin Samuel | Monoclonal antibody |
-
1983
- 1983-02-25 GB GB08305364A patent/GB2117789B/en not_active Expired
- 1983-03-18 JP JP58501140A patent/JPS59500633A/en active Granted
- 1983-03-18 EP EP19830901088 patent/EP0103620B1/en not_active Expired
- 1983-03-18 WO PCT/GB1983/000081 patent/WO1983003477A1/en not_active Ceased
- 1983-03-18 DE DE8383901088T patent/DE3377745D1/en not_active Expired
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1981002104A1 (en) * | 1980-01-28 | 1981-08-06 | S Svensson | Isolation of carbohydrates from cell tissue |
| WO1982003089A1 (en) * | 1981-03-06 | 1982-09-16 | Lennox Edwin Samuel | Monoclonal antibody |
Non-Patent Citations (8)
| Title |
|---|
| Biological Abstracts, Vol. 72, No. 2, published in 1981, (Philadelphia, US) D. VOAK et al.: "Monoclonal Anti-A from a Hybrid Mycloma: Evaluation as a Blood Grouping Reagent", see page 1034, column 2, Abstract No. 9903, Vox Sang. 39(3), 1980, 134-140 * |
| CHEMICAL ABSTRACTS, Vol. 97, No. 11, 13 September 1982 (Columbus, Ohio, US) D. VOAK et al.: "Monoclonal Anti-A and Anti-B: Development as Cost Effective Reagents", see page 587, column 2, Abstract No. 90128c, Med. Lab. Sci. 1982, 39(2), 109-22 * |
| Clinical Chemistry, Vol. 27, No. 11, published in November 1981 (Easton, US) E.D. SEVIER et al.: "Monoclonal Antibodies in Clinical Immunology", see pages 1797-1806 * |
| Medical Laboratory Sciences, Vol. 36, published in 1979, A.D. BLANN: "Cell Hybrids: an Important New Source of Antibody Production", see pages 329-338 * |
| SACKS ET AL., VOX. SANG., vol. 40, 1981, pages 99 - 104 |
| The Journal of Immunology, Vol. 129, No. 2 published in August, 1982 (Baltimore, US) D.R. BUNDLE et al.: "Hybridomas Specific for Carbohydrates; Synthetic Human Blood Group Antigens for the Production, Selection and Characterization of Monoclonal Typing Regents", see pages 678-682 * |
| VOAK ET AL., VOX. SANG., vol. 39, 1980, pages 134 - 140 |
| Vox Sanguinis, Vol. 40, published in 1981, S.H. SARKS et al.: "Monoclonal anti-B as a New Blood-Typing Reagent", see pages 99-104 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0115476A3 (en) * | 1983-01-21 | 1988-10-12 | Monocarb Ab | Monoclonal antibody formulation for diagnostic use |
| EP0138377A1 (en) * | 1983-09-16 | 1985-04-24 | The Wistar Institute | Lewis blood group phenotype assay |
| US4764465A (en) * | 1984-04-26 | 1988-08-16 | Cetus Corporation | Human monoclonal antibody against group A red blood cells |
| EP0223978A1 (en) * | 1985-10-11 | 1987-06-03 | Abbott Laboratories | Diagnostic test |
| EP0232706A3 (en) * | 1986-01-10 | 1988-11-30 | Sloan-Kettering Institute For Cancer Research | Blood group antigen panel |
| US5583003A (en) * | 1989-09-25 | 1996-12-10 | Agen Limited | Agglutination assay |
| RU2138812C1 (en) * | 1998-11-11 | 1999-09-27 | Сахаров Роман Сергеевич | METHOD OF PREPARING SPECIFICALLY REACTING IN REACTION ABSORPTION-ELUTION SERA ANTI-A (α), ANTI-B (β) IN DETECTION OF ANTIGENS A, B IN HUMAN EXCRETION TRACES |
| CN102944684A (en) * | 2012-11-07 | 2013-02-27 | 武汉康莱博生物科技有限公司 | A and B-type blood type resistant sizing reagent |
| CN102944684B (en) * | 2012-11-07 | 2014-09-10 | 武汉康莱博生物科技有限公司 | A and B-type blood type resistant sizing reagent |
| WO2020106623A1 (en) | 2018-11-19 | 2020-05-28 | New Health Sciences, Inc. | Methods for the preservation of reagent red blood cells using carbon monoxide |
| WO2021046424A2 (en) | 2019-09-05 | 2021-03-11 | Hemanext Inc. | Methods for the preservation of reagent red blood cells using carbon monoxide |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0103620A1 (en) | 1984-03-28 |
| GB8305364D0 (en) | 1983-03-30 |
| DE3377745D1 (en) | 1988-09-22 |
| JPS59500633A (en) | 1984-04-12 |
| EP0103620B1 (en) | 1988-08-17 |
| JPH0314139B2 (en) | 1991-02-26 |
| GB2117789A (en) | 1983-10-19 |
| GB2117789B (en) | 1985-08-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Carrel et al. | Subsets of human Ia-like molecules defined by monoclonal antibodies | |
| Brandtzaeg | Human secretory immunoglobulin M. An immunochemical and immunohistochemical study | |
| EP0043718B1 (en) | Improvements in or relating to cell lines | |
| Svejgaard et al. | Cross-reactive human HL-A isoantibodies | |
| Lachmann et al. | Three rat monoclonal antibodies to human C3 | |
| DE3486356T2 (en) | MONOCLONAL ANTIBODIES AGAINST ONCOPROTEINS INDUCED BY POLYPEPTIDES. | |
| EP0014519A2 (en) | Cell lines, process for preparing them and process for producing antibodies | |
| JPH0159871B2 (en) | ||
| WO1982001461A1 (en) | Human hybridomas,precursors and products | |
| Campbell et al. | Removal of immunoglobulin-bearing lymphocytes by anti-immunoglobulin-coated columns | |
| EP0103620B1 (en) | Blood cell grouping reagent | |
| DE3218312A1 (en) | MONOCLONAL ANTIBODIES, HYBRIDOMA CELL CLONES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE FOR DETECTING BREAST CANCER AND MALIGNER LYMPHOGRANULOMATOSIS | |
| Vyas et al. | Lmmunobiology of human anti‐lgA: a serologic and immunogenetic study of immunization to IgA in transfusion and pregnancy | |
| US5081230A (en) | Monoclonal antibodies reactive with normal and oncogenic forms of the ras p21 protein | |
| Rudich et al. | Human B cell activation. Evidence for diverse signals provided by various monoclonal anti-IgM antibodies. | |
| EP0576093B1 (en) | Human monoclonal anti-Rhesus (D) antibodies and cell lines producing same | |
| Zola et al. | Monoclonal antibodies against antigens of the human platelet surface: preparation and properties | |
| US4818689A (en) | Late differentiation antigen associated with helper T lymphocyte function | |
| US5330896A (en) | Monoclonal antibodies to an autocrine growth factor antigen that binds to activated lymphocytes and cancer cells | |
| Luo et al. | A novel monoclonal antibody based diagnostic test for alpha-thalassemia-1 carriers due to the (-SEA/) deletion | |
| Fine et al. | Waldenström's macroglobulinemia in monozygotic twins | |
| EP0378572A1 (en) | HUMAN ANTI-Rh(D) MONOCLONAL ANTIBODIES | |
| AU636867B2 (en) | Rat monoclonal antibodies directed against human antigens and processes for preparation thereof | |
| AU615233B2 (en) | Monoclonal antibody that recognizes an epitope of the gamma chain of human t cell surface receptors | |
| Sanders et al. | Monoclonal antibody to chicken fetal antigens on normal erythroid cells and hematopoietic-lymphoid tumor cell lines |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Designated state(s): JP US |
|
| AL | Designated countries for regional patents |
Designated state(s): AT BE CH DE FR LU NL SE |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 1983901088 Country of ref document: EP |
|
| WWP | Wipo information: published in national office |
Ref document number: 1983901088 Country of ref document: EP |
|
| WWG | Wipo information: grant in national office |
Ref document number: 1983901088 Country of ref document: EP |