US4275053A
Expired / Lapsed
US
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Blood cell typing and compatibility test procedure
Assignee: Icahn School of Medicine at Mount Sinai
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Filing: 1978-06-14
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Granted: 1981-06-23
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Expires: 1998-06-14
Abstract
Abstract A solid-phase blood typing procedure is described based upon either aggluation or immune lysis. In this invention, a monolayer of cells is irreversibly bound to a solid matrix, and thereafter a serum containing antibodies is brought into contact with the bound cell layer. Immuno-adsorption of antibodies by the bound cells occurs where the antigens of the cell membranes and the antibodies in the serum are complementary to each other. This antibody-sensitized monolayer of blood cells can either bind a second layer of blood cells carrying complementary antigen (solid-phase agglutination) or undergo lysis in the presence of serum lytic complement (solid-phase immune lysis). Carrying out these reactions with a monolayer of blood cells bound to a solid matrix allows quantitative evaluation of results by such standard instrumentable procedures as densitometric scanning, radioisotope counting, etc.
Inventors
- 👤 Richard E. Rosenfield
- 👤 Shaul Kochwa
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 15:50
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/80; Y; Y10; Y10S; Y10S436/00; Y10S436/821
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
112 words
A method for assaying cell type or compatibility on a solid matrix comprising the steps of (a) forming a first monolayer of cells irreversibly bound to a solid matrix, said cells having intrinsic or acquired antigens thereon; (b) contacting said layer (a) with a solution containing antibodies, whereby a layer of antibodies is bound by immunoadsorption, to the extent that the antibodies of said solution are reactive with the antigens of the cells of said first layer; (c) thereafter applying a suspension of second cells, said second cells having antigens thereon; and (d) subsequently measuring the extent of immunoadherence of said second cells by antibody bound to said first layer of cells.
Claim 2
Independent Claim
112 words
A method for assaying cell type or compatibility on a solid matrix comprising the steps of (a) forming a first monolayer of cells irreversibly bound to a solid matrix, said cells having intrinsic or acquired antigens thereon; (b) contacting said layer (a) with a solution containing antibodies, whereby a layer of antibodies is bound by immunoadsorption, to the extent that the antibodies of said solution are reactive with the antigens of the cells of said first layer; (c) thereafter applying a suspension of second cells, said second cells having antigens thereon; and (d) subsequently measuring the extent of immunoadherence of said second cells by antibody bound to said first layer of cells.
Claim 3
Dependent Claim
34 words
A method according to claim 1 wherein said first layer of cells and said second cells are the cells of a first person and said solution is a serum obtained from a second person.
Claim 4
Dependent Claim
52 words
A method according to claim 1 wherein layer (c) is formed by applying cells from a cell population to be tested in a number essentially just sufficient to form a monolayer, and the extent of immunoadherence of said monolayer is a function of the percentage of tested cells that carry specific antigen.
Claim 5
Dependent Claim
56 words
A method according to claim 1 wherein said solution containing antibodies is a mixture of (i) a solution of an antibody reactive by immunoadsorption with an antigen carried by the cells of said layer (a) and (ii) a second solution or suspension to be assayed for antigenic determinants by inhibition of antibody in said solution (i).
Claim 6
Dependent Claim
83 words
The method according to claim 1 wherein the antigens of said first cell layer (a) are antigens carried by immunoglobulins or complement components bound on the surfaces of said cell population; the antibodies in said solution used in step (b) are anti-immunoglobulin or anti-complement to the antigenic determinants carried by the immunoglobulins or complement components to be assayed on said first cell monolayer; and the cells of said second layer carry antigenic determinants which are immunoadherent to the antibodies employed in step (b).
Claim 7
Dependent Claim
29 words
The method according to claim 5, wherein the immunoglobulins or complement components are bound to the cells of said cell layer (a) after the layer (a) has been constructed.
Claim 8
Dependent Claim
26 words
The method according to claim 5, wherein the cells bound in step (a) carry or are suspected to carry immunoglobulins or complement components to be assayed.
Claim 9
Dependent Claim
73 words
The method according to claim 6 wherein the layer (a) is a monolayer of platelets constructed by: (i) treating a solid matrix of polystyrene successively with a fibrinogen solution and a polylysine solution; (ii) treating a suspension of platelets with an acetylating agent and washing the acetylated platelets; and (iii) applying the washed acetylated platelets to the fibrinogen-polylysine-treated substrate under conditions effective to form a monolayer of platelets irreversibly bound to the substrate.
Claim 10
Dependent Claim
28 words
The method according to claim 8 wherein the immunoglobulins or complement components are bound to the platelets of cell layer (a) after the cell layer has been constructed.
Claim 11
Dependent Claim
28 words
The method according to claim 8, wherein the platelets treated with an acetylating agent carry or are suspected to carry an immunoglobulin or complement components to be assayed.
Claim 12
Dependent Claim
46 words
The method according to claim 1 wherein the cell monolayer (a) is formed by applying a suspension of said cells to said solid matrix to be coated, there being, on the surface of said matrix, reactive chemical groups effective to couple said cells to said matrix.
Claim 13
Dependent Claim
45 words
The method according to claim 11 wherein said reactive chemical groups are formed by successively treating a solid matrix of a polystyrene with a fibrinogen solution and a polysine solution under conditions effective to form said reactive chemical groups on the surface of said matrix.
Claim 14
Independent Claim
112 words
A method for assaying cell type or compatibility on a solid matrix comprising the steps of (a) forming a first monolayer of erythrocytes irreversibly bound to a solid matrix, said erythrocytes having intrinsic or acquired antigens thereon; (b) contacting said layer (a) with a solution containing antibodies, whereby a layer of antibodies is bound by immunoadsorption to the extent that the antibodies of said solution are reactive with the antigens of the erythrocytes of said first layer; (c) thereafter applying a suspension of second erythrocytes, said second erythrocytes having antigens thereon; and (d) subsequently measuring the extent of immunoadherence of said second erythrocytes by antibody bound to said first layer of erythrocytes.
Claim 15
Independent Claim
112 words
A method for assaying cell type or compatibility on a solid matrix comprising the steps of (a) forming a first monolayer of erythrocytes irreversibly bound to a solid matrix, said erythrocytes having intrinsic or acquired antigens thereon; (b) contacting said layer (a) with a solution containing antibodies, whereby a layer of antibodies is bound by immunoadsorption to the extent that the antibodies of said solution are reactive with the antigens of the erythrocytes of said first layer; (c) thereafter applying a suspension of second erythrocytes, said second erythrocytes having antigens thereon; and (d) subsequently measuring the extent of immunoadherence of said second erythrocytes by antibody bound to said first layer of erythrocytes.
Claim 16
Dependent Claim
21 words
The method according to claim 13 wherein said first layer of erythrocytes is lysed prior to application of the second erythrocytes.
Claim 17
Dependent Claim
21 words
The method according to claim 14 wherein the erythrocytes of said first layer are lysed after application of said solution (b).
Claim 18
Dependent Claim
34 words
A method according to claim 13 wherein said first layer of erythrocytes and said second erythrocytes are the erythrocytes of a first person and said solution is a serum obtained from a second person.
Claim 19
Dependent Claim
53 words
A method according to claim 13 wherein layer (c) is formed by applying cells from an erythrocyte population to be tested in a number essentially just sufficient to form a monolayer, and the extent of immunoadsorption of said second monolayer is a function of the percentage of tested erythrocytes that carry specific antigen.
Claim 20
Dependent Claim
56 words
A method according to claim 13 wherein said solution containing antibodies is a mixture of (i) a solution of an antibody reactive by immunoadsorption with an antigen carried by the erythrocytes of said layer (a), and (ii) a second solution of suspension to be assayed for antigenic determinants by inhibition of antibody in said solution (i).
Claim 21
Dependent Claim
81 words
The method according to claim 13 wherein the antigens of said first erythrocyte layer are antigens carried by immunoglobulins or complement components bound on the surfaces of said erythrocyte population, the antibodies in said solution used in step (b) are anti-immunoglobulin or anti-complement to the antigenic determinants carried by the antibody or complement to be assayed on said first erythrocyte monolayer, and the erythrocytes of said second layer carry antigenic determinants which are immunoadherent to the antibodies employed in step (b).
Claim 22
Dependent Claim
28 words
The method according to claim 19 wherein the immunoglobulins or complement components are bound to the erythrocytes of said layer (a) after said layer (a) has been constructed.
Claim 23
Dependent Claim
46 words
The method according to claim 13 wherein the erythrocyte monolayer (a) is formed by applying a suspension of said erythrocytes to said solid matrix to be coated, there being, on the surface of said matrix, reactive chemical groups effective to couple said erythrocytes to said matrix.
Claim 24
Dependent Claim
45 words
The method according to claim 21 wherein said reactive chemical groups are formed by successively treating a solid matrix of a polystyrene with a fibrinogen solution and a polylysine solution under conditions effective to form said reactive chemical groups on the surface of said matrix.
Claim 25
Independent Claim
189 words
A method for assaying cell type or compatibility on a solid matrix comprising the steps of (a) forming a first monolayer of washed erythrocytes irreversibly bound to a solid matrix, said erythrocytes having intrinsic or acquired antigens thereon; (b) contacting said layer (a) with a solution containing antibodies under conditions of reduced ionic concentration, whereby a layer of antibodies is bound by immunoadsorption to said first monolayer of erythrocytes to the extent that the antibodies of said solution are reactive with the antigens of the erythrocytes of said first layer; (c) thereafter applying a suspension of second erythrocytes, said second erythrocytes having antigens thereon, and allowing said second erythrocytes to settle on said solid matrix as a second monolayer over said first layer of erythrocytes; (d) after the said second monolayer has formed, adding protamine sulfate to said second suspension in an amount sufficient to promote adherence of said second erythrocytes to antibodies bound to said first monolayer; and (e) thereafter washing to remove second erythrocytes which are not immunobound, and measuring the extent of immunoadherence of said second erythrocytes to antibodies bound to said first layer of erythrocytes.
Claim 26
Independent Claim
189 words
A method for assaying cell type or compatibility on a solid matrix comprising the steps of (a) forming a first monolayer of washed erythrocytes irreversibly bound to a solid matrix, said erythrocytes having intrinsic or acquired antigens thereon; (b) contacting said layer (a) with a solution containing antibodies under conditions of reduced ionic concentration, whereby a layer of antibodies is bound by immunoadsorption to said first monolayer of erythrocytes to the extent that the antibodies of said solution are reactive with the antigens of the erythrocytes of said first layer; (c) thereafter applying a suspension of second erythrocytes, said second erythrocytes having antigens thereon, and allowing said second erythrocytes to settle on said solid matrix as a second monolayer over said first layer of erythrocytes; (d) after the said second monolayer has formed, adding protamine sulfate to said second suspension in an amount sufficient to promote adherence of said second erythrocytes to antibodies bound to said first monolayer; and (e) thereafter washing to remove second erythrocytes which are not immunobound, and measuring the extent of immunoadherence of said second erythrocytes to antibodies bound to said first layer of erythrocytes.
Claim 27
Dependent Claim
30 words
The method according to claim 23, wherein between steps (a) and (b) and between steps (b) and (c), the first monolayer is washed to remove nonadherent cells and unwanted reagents.
Claim 28
Dependent Claim
21 words
The method according to claim 23, wherein following step (b) said first monolayer of erythrocytes is hypotonically lysed with distilled water.
Claim 29
Dependent Claim
34 words
A method according to claim 23, wherein said solution containing antibodies used in step (b) is a serum of antibody diluted at least 1:2 with a 1:9% glycine solution at a pH of 7.0.
Claim 30
Dependent Claim
30 words
The method according to claim 23, wherein said substrate in step (e) is washed with a solution consisting essentially of isotonic NaCl and phosphate buffer at a pH of 3.
Claim 31
Independent Claim
125 words
A method for assaying cell type or compatibility on a solid matrix comprising the steps of (a) forming a first monolayer of cells irreversibly bound to a solid matrix, said cells having or carrying antigens thereon; (b) contacting said layer (a) with a solution containing antibodies capable of forming a layer of antibodies bound by immunoadsorption to said first layer of cells to the extent that the antibodies of said solution are reactive with the antigens of the cells of said layer; and (c) thereafter measuring the extent of immunoadsorption of said antibodies by applying a second solution containing lytic complement effective to lyse the cells of said layer (a) in the presence of immunoadsorbed antibody and observing the extent of immune lysis which occurs.
Claim 32
Independent Claim
125 words
A method for assaying cell type or compatibility on a solid matrix comprising the steps of (a) forming a first monolayer of cells irreversibly bound to a solid matrix, said cells having or carrying antigens thereon; (b) contacting said layer (a) with a solution containing antibodies capable of forming a layer of antibodies bound by immunoadsorption to said first layer of cells to the extent that the antibodies of said solution are reactive with the antigens of the cells of said layer; and (c) thereafter measuring the extent of immunoadsorption of said antibodies by applying a second solution containing lytic complement effective to lyse the cells of said layer (a) in the presence of immunoadsorbed antibody and observing the extent of immune lysis which occurs.
Claim 33
Dependent Claim
32 words
A method according to claim 28, wherein the cells of said first layer are cells of a first person and said solution containing antibodies is a serum obtained from a second person.
Claim 34
Dependent Claim
45 words
The method according to claim 28, wherein said cell layer (a) is formed by applying a suspension of cells to said solid matrix to be coated, there being, on the surface of said matrix, reactive chemical groups effective to couple said cells to said matrix.
Claim 35
Dependent Claim
15 words
The method according to claim 30, wherein the cells of said first layer are erythrocytes.
Claim 36
Dependent Claim
45 words
The method according to claim 30, wherein said reactive chemical groups are formed by successively treating a solid matrix of a polystyrene with a fibrinogen solution and a polylysine solution under conditions effective to form said reactive chemical groups on the surface of said matrix.
Claim 37
Dependent Claim
46 words
The method according to claim 29, wherein the cell monolayer (a) is formed by applying a suspension of said cells to said solid matrix to be coated, there being, on the surface of said matrix, reactive chemical groups effective to couple said cells to said matrix.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1984-06-22 | 1984-12-22 | 1985-06-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-06-22 | 1988-12-22 | 1989-06-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-06-22 | 1992-12-22 | 1993-06-23 | $7700.00 | Window Closed / Lapsed |