Receptors specific for hapten-modified self proteins

Claim 2

An epitope-specific reagent comprising a receptor raised in a host animal to an immunogenic, modified protein; said immunogenic, modified protein comprising a reduced glycosylated or alkylated free amine-modified plasma protein that when native and unmodified is (a) homologous or autologous to said host animal and (b) non-immunogenic in said host animal, said receptor binding to said reduced glycosylated or alkylated free amine-modified protein but not to said unmodified native protein.

Claim 3

The epitope-specific reagent according to claim 1 wherein said receptor is a monoclonal antibody.

Claim 4

The epitope-specific reagent according to claim 1 wherein said receptor is a monoclonal antibody produced and secreted by a hybridoma selected from the group consisting of hybridomas ATCC HB 8354, HB 8355, HB 8356, HB 8357, HB 8358 and HB 8359.

Claim 5

The epitope-specific reagent according to claim 1 wherein said receptor is in substantially pure form.

Claim 6

The epitope-specific reagent according to claim 1 wherein the immunogenic, modified protein is a reduced glycosylated plasma protein.

Claim 7

The epitope-specific reagent according to claim 1 wherein the native unmodified protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 10

The method according to claim 7 wherein the immunogenic, reduced glycosylated or alkylated free amine-modified plasma protein is a reduced glycosylated plasma protein.

Claim 11

The method according to claim 7 wherein the protein in unmodified form is a native plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 14

The method according to claim 10 wherein said receptor is in substantially pure form.

Claim 15

The method according to claim 10 further including the step of administering to said host, after step (a) but before step (b), a second immunogenic plasma protein in a reduced glycosylated or alkylated free amine-modified form in an amount sufficient to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with the epitope of said first-named reduced glycosylated or alkylated free amine-modified protein, the second protein being immunogenic in the host in an unmodified native form.

Claim 16

The method according to claim 10 wherein the epitope-specific reagent is a monoclonal antibody produced and secreted by a hybridoma selected from the group consisting of hybridomas ATCC HB 8355, HB 8356, HB 8357, HB 8359, HB 8358 and HB 8354.

Claim 17

The method according to claim 10 wherein the reduced glycosylated or alkylated free amine-modified form of the immunogenic protein is a reduced glycosylated plasma protein.

Claim 18

The method according to claim 10 wherein the protein in unmodified form is a native plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 19

A monoclonal antibody which immunoreacts with a reduced glycosylated free amine-modified plasma protein but does not immunoreact with the non-reduced non-glycosylated native protein prepared by the method comprising the steps of: (a) administering to a host a reduced glucosylated free amine-modified plasma protein in an amount sufficient to induce the production of antibodies to the reduced glycosylated free amine-modified plasma protein, said non-reduced non-glycosylated native protein being non-immunogenic in the host; (b) recovering the antibody producing cells from the host; (c) forming cell hybrids by fusing said antibody producing cells to myeloma cells; (d) culturing said hybrids; and (e) collecting said monoclonal antibody as a product of said hybrids.

Claim 20

A monoclonal antibody which immunoreacts with a reduced glycosylated free amine-modified plasma protein but does not immunoreact with the non-reduced non-glycosylated native protein prepared by the method comprising the steps of: (a) administering to a host a reduced glucosylated free amine-modified plasma protein in an amount sufficient to induce the production of antibodies to the reduced glycosylated free amine-modified plasma protein, said non-reduced non-glycosylated native protein being non-immunogenic in the host; (b) recovering the antibody producing cells from the host; (c) forming cell hybrids by fusing said antibody producing cells to myeloma cells; (d) culturing said hybrids; and (e) collecting said monoclonal antibody as a product of said hybrids.

Claim 23

The method according to claim 17 wherein said non-reduced non-glucosylated native protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 24

The monoclonal antibody prepared by the method of claim 17.

Claim 27

The method according to claim 20 wherein said non-reduced non-glucosylated protein native is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 28

The monoclonal antibody prepared by the method of claim 20.

Claim 31

The method according to claim 23 wherein said non-reduced non-glucosylated protein native is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 32

The monoclonal antibody prepared by the method of claim 23.

Claim 35

The method according to claim 26 wherein said non-reduced non-glycosylated native protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 36

The monoclonal antibody prepared by the method of claim 26.

Claim 39

The method according to claim 29 whrein said non-reduced non-glucosylated native protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 40

The monoclonal antibody prepared by the method of claim 29.

Claim 43

The method according to claim 32 wherein said non-reduced non-glucosylated native protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 44

The monoclonal antibody prepared by the method of claim 32.

Claim 47

The method according to claim 35 wherein said non-reduced non-glucosylated native protein plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 48

The monoclonal antibody prepared by the method of claim 35.

Claim 51

The method according to claim 38 wherein said non-reduced non-glucosylated native protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 52

The monoclonal antibody prepared by the method of claim 38.

Claim 55

The method according to claim 41 wherein said non-reduced non-glucosylated native protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 56

The monoclonal antibody prepared by the method of claim 41.

Claim 59

The method according to claim 44 wherein said non-reduced non-glucosylated native protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 60

The monoclonal antibody prepared by the method of claim 44.

Claim 63

The method according to claim 47 wherein said non-reduced non-glucosylated native protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 64

The monoclonal antibody prepared by the method of claim 47.

Claim 67

The method according to claim 50 wherein said non-reduced non-glucosylated native protein is a plasma protein selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 68

The monoclonal antibody prepared by the method of claim 50.

Claim 69

A diagnostic system for assaying for the presence of an antigenic reduced glycosylated or alkylated free amine-modified protein, said system in kit form including in at least one package as an active ingredient an effective amount of an epitope-specific reagent which when introduced into a sample reacts with an antigenic, reduced glycosylated or alkylated free amine-modified protein but does not react with an unmodified native protein as a measure of the reduced glycosylated or alkylated free amine-modified protein in the sample.

Claim 70

A diagnostic system for assaying for the presence of an antigenic reduced glycosylated or alkylated free amine-modified protein, said system in kit form including in at least one package as an active ingredient an effective amount of an epitope-specific reagent which when introduced into a sample reacts with an antigenic, reduced glycosylated or alkylated free amine-modified protein but does not react with an unmodified native protein as a measure of the reduced glycosylated or alkylated free amine-modified protein in the sample.

Claim 71

The diagnostic system according to claim 53 further including a second package including a label for identifying the presence of said unmodified protein.

Claim 72

The diagnostic system according to claim 53 wherein the epitope-specific reagent is a monoclonal antibody.

Claim 73

The diagnostic system according to claim 53 wherein said reduced glycosylated or alkylated free amine-modified protein is a reduced glycosylated plasma protein.

Claim 76

An assay for determining the presence and amount of an antigenic, reduced glycosylated or alkylated free amine-modified protein comprising the steps of: (a) providing a first receptor covalently attached in a known amount to a solid support, the first receptor binding to a first epitope of a protein to be assayed when the protein is in reduced glycosylated or alkylated free amine-modified and unmodified native forms; (b) providing a second receptor that binds to a second epitope of the protein to be assayed when that protein is in reduced glycosylated or alkylated free amine-modified or unmodified native forms; (c) providing an epitope-specific reagent comprising a receptor raised in a host animal to an immunogenic, modified protein, said immunogenic, modified protein comprising a reduced glycosylated or alkylated free amine-modified plasma protein that when in unmodified native form is (i) homologous or autologous to said host animal and (ii) non-immunogenic in said host animal, said receptor binding to said reduced glycosylated or alkylated free amine-modified plasma protein but not to said unmodified native protein, said epitope-specific reagent binding to a third epitope of the protein that is present only when the protein is in reduced glycosylated or alkylated free amine-modified form; (d) admixing a predetermined amount of said covalently attached first receptor and a predetermined amount of a sample to be assayed (i) for the presence of antigenic, reduced glycosylated or alkylated free amine-modified protein to which the epitope-specific reagent binds, and (ii) for the presence of the unmodified native protein to which the first and the second receptors bind; (e) maintaining that admixture for a period of time sufficient for said covalently attached first receptor to bind reduced glycosylated or alkylated free amine-modified and unmodified native proteins present and form binding complexes therewith; (f) separating the formed binding complexes from the remainder of the admixture; (g) dividing the formed binding complexes into at least a first aliquot and a second aliquot; (h) admixing the first aliquot with a predetermined amount of the second receptor and maintaining that admixture for a period of time sufficient to form second binding complexes between the second receptor and the first-formed binding complexes; (i) determining the amount of second binding complexes formed to thereby determine the total amount of both reduced glycosylated or alkylated free amine-modified and unmodified protein present in the sample; (j) admixing the second aliquot with a predetermined amount of the epitope-specific reagent and maintaining that admixture for a period of time sufficient to form a third binding complex between the eiptope-specific reagent and reduced glycosylated or alkylated free amine-modified protein present in the first-formed binding complexes; and (k) determining the amount of third binding complex formed to thereby determine the amount of reduced glycosylated or alkylated free amine-modified protein present in the sample.

Claim 77

An assay for determining the presence and amount of an antigenic, reduced glycosylated or alkylated free amine-modified protein comprising the steps of: (a) providing a first receptor covalently attached in a known amount to a solid support, the first receptor binding to a first epitope of a protein to be assayed when the protein is in reduced glycosylated or alkylated free amine-modified and unmodified native forms; (b) providing a second receptor that binds to a second epitope of the protein to be assayed when that protein is in reduced glycosylated or alkylated free amine-modified or unmodified native forms; (c) providing an epitope-specific reagent comprising a receptor raised in a host animal to an immunogenic, modified protein, said immunogenic, modified protein comprising a reduced glycosylated or alkylated free amine-modified plasma protein that when in unmodified native form is (i) homologous or autologous to said host animal and (ii) non-immunogenic in said host animal, said receptor binding to said reduced glycosylated or alkylated free amine-modified plasma protein but not to said unmodified native protein, said epitope-specific reagent binding to a third epitope of the protein that is present only when the protein is in reduced glycosylated or alkylated free amine-modified form; (d) admixing a predetermined amount of said covalently attached first receptor and a predetermined amount of a sample to be assayed (i) for the presence of antigenic, reduced glycosylated or alkylated free amine-modified protein to which the epitope-specific reagent binds, and (ii) for the presence of the unmodified native protein to which the first and the second receptors bind; (e) maintaining that admixture for a period of time sufficient for said covalently attached first receptor to bind reduced glycosylated or alkylated free amine-modified and unmodified native proteins present and form binding complexes therewith; (f) separating the formed binding complexes from the remainder of the admixture; (g) dividing the formed binding complexes into at least a first aliquot and a second aliquot; (h) admixing the first aliquot with a predetermined amount of the second receptor and maintaining that admixture for a period of time sufficient to form second binding complexes between the second receptor and the first-formed binding complexes; (i) determining the amount of second binding complexes formed to thereby determine the total amount of both reduced glycosylated or alkylated free amine-modified and unmodified protein present in the sample; (j) admixing the second aliquot with a predetermined amount of the epitope-specific reagent and maintaining that admixture for a period of time sufficient to form a third binding complex between the eiptope-specific reagent and reduced glycosylated or alkylated free amine-modified protein present in the first-formed binding complexes; and (k) determining the amount of third binding complex formed to thereby determine the amount of reduced glycosylated or alkylated free amine-modified protein present in the sample.

Claim 78

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced free amine-glucosylted plasma protein, said antibody reacting with the human reduced glucosylated free amine-modified plasma protein, glucitollysine, glucitolpolylysine, arginine and lysine, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glucosylated plasma protein, a non-reduced non-glucosylated native protein, methionine, sorbitol, manitol, glutamine or glucose.

Claim 79

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced free amine-glucosylted plasma protein, said antibody reacting with the human reduced glucosylated free amine-modified plasma protein, glucitollysine, glucitolpolylysine, arginine and lysine, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glucosylated plasma protein, a non-reduced non-glucosylated native protein, methionine, sorbitol, manitol, glutamine or glucose.

Claim 80

The monoclonal antibody according to claim 59 which is of the class IgG.

Claim 81

The monoclonal antibody according to claim 59 which is produced and secreted by a hybridoma formed by fusion of P3×63Ag8 myeloma cells and spleen cells from a Balb/c mouse.

Claim 82

The monoclonal antibody according to claim 59 wherein said murine reduced glucosylated free amine-modified plasma protein and said human rediced glucosylated free amine-modified plasma protein when in non-reduced and non-glucosylated native forms are selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 83

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced glucosylated free amine-plasma protein, said antibody reacting with the human reduced glucosylated free-amine-modified plasma protein, glucitollysine, glucitolpolylysine, arginine, lysine, methionine, sorbitol and mannitol, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glucosylated plasma protein, a non-reduced non-glucosylated native plasma protein, glutamine or glucose.

Claim 84

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced glucosylated free amine-plasma protein, said antibody reacting with the human reduced glucosylated free-amine-modified plasma protein, glucitollysine, glucitolpolylysine, arginine, lysine, methionine, sorbitol and mannitol, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glucosylated plasma protein, a non-reduced non-glucosylated native plasma protein, glutamine or glucose.

Claim 85

The monoclonal antibody according to claim 63 which is of the class IgG.

Claim 86

The monoclonal antibody according to claim 63 which is produced and secreted by a hybridoma formed by fusion of P3×63Ag8 myeloma cells and spleen cells fron a Balb/c mouse.

Claim 87

The monoclonal antibody according to claim 63 wherein said murine reduced glucosylated free amine-modified plasma protein and said human reduced glucosylated free amine-modified plasma protein when in non-reduced and non-glucosylated native forms are selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 88

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced glucosylated free amine-modified plasma protein, said antibody reacting with the human reduced glucosylated free amine-modified plasma protein, glucitollysine, glucitolpolylysine and arginine, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glucosylated plasma protein, a non-reduced non-glucosylated native plasma protein, lysine, methionine, sorbitol, mannitol, glutamine or glucose.

Claim 89

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced glucosylated free amine-modified plasma protein, said antibody reacting with the human reduced glucosylated free amine-modified plasma protein, glucitollysine, glucitolpolylysine and arginine, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glucosylated plasma protein, a non-reduced non-glucosylated native plasma protein, lysine, methionine, sorbitol, mannitol, glutamine or glucose.

Claim 90

The monoclonal antibody according to claim 67 which is of the class IgG.

Claim 91

The monoclonal antibody according to claim 67 which is produced and secreted by a hybridoma formed by fusion of P3×63Ag8 myeloma cells and spleen cells from a Balb/c mouse.

Claim 92

The monoclonal antibody according to claim 67 wherein said murine reduced glucosylated free amine-modified plasma protein and said human reduced glucosylated free amine-modified plasma protein when in non-reduced and non-glucosylated native forms are selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 93

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced glucosylated free amine-modified plasma protein, said antibody reacting with the human reduced glucosylated free amine-modified plasma protein, glucitollysine, glucitolpolylysine, lysine, methionine, sorbitol and glutamine, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glycosylated protein, a non-reduced non-glucosylated native plasma protein, arginine, mannitol or glucose.

Claim 94

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced glucosylated free amine-modified plasma protein, said antibody reacting with the human reduced glucosylated free amine-modified plasma protein, glucitollysine, glucitolpolylysine, lysine, methionine, sorbitol and glutamine, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glycosylated protein, a non-reduced non-glucosylated native plasma protein, arginine, mannitol or glucose.

Claim 95

The monoclonal antibody according to claim 71 which is of the class IgG.

Claim 96

The monoclonal antibody according to claim 71 which is produced and secreted a hybridoma formed by fusion of P3×63Ag8 myeloma cells and spleed cells fropm a Balb/c mouse.

Claim 97

The monoclonal antibody according to claim 71 wherein said murine reduced glucosylated free amine-modified plasma protein and said human reduced glucosylated free amine-modified plasma protein when in non-reduced and non-glucosylated native forms are selected from the group consisting of a lipoprotein, hemoglobin, albumin and tranferrin.

Claim 98

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced glucosylated free amine-modified plasma protein, said antibody reacting with the human reduced glucosylated free amine-modified plasma protein glucitollysine, glucitolpolylysine and sorbitol, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glucosylated plasma protein, a non-reduced non-glucosylated native plasma protein, arginine, lysine, methionine, mannitol, glutamine or glucose.

Claim 99

A monoclonal antibody produced and secreted by a hybridoma formed by fusion of a cell from a mouse myeloma cell line and a spleen cell from a mouse previously immunized first with a murine reduced glucosylated free amine-modified plasma protein and then boosted with a human reduced glucosylated free amine-modified plasma protein to enhance the proliferative expansion and differentiation of B cell clones producing antibodies capable of crossreacting with said murine reduced glucosylated free amine-modified plasma protein, said antibody reacting with the human reduced glucosylated free amine-modified plasma protein glucitollysine, glucitolpolylysine and sorbitol, but not with a non-reduced glucosylated free amine-modified plasma protein, a reduced non-glucosylated plasma protein, a non-reduced non-glucosylated native plasma protein, arginine, lysine, methionine, mannitol, glutamine or glucose.

Claim 100

The monoclonal antibody according to claim 75 which is of the class IgM.

Claim 101

The monoclonal antibody according to claim 75 which is produced and secreted by a hybridoma formed by fusion of P3×63Ag8 myeloma cells and spleen cells from a Balb/c mouse.

Claim 102

The monoclonal antibody according to claim 75 wherein said murine reduced glucosylated free amine-modified plasma protein and said human reduced glucosylated free amine-modified plasma protein when in non-reduced and non-glucosylated native forms are selected from the group consisting of a lipoprotein, hemoglobin, albumin and transferrin.

Claim 103

A hybridoma having ATCC accession number HB 8355.

Claim 104

A hybridoma having ATCC accession number HB 8355.

Claim 105

The monoclonal receptor that reacts with glucilollysine and is produced and secreted by the hybridoma of claim 79.

Claim 106

A hybridoma having ATCC accession number HB 8356.

Claim 107

A hybridoma having ATCC accession number HB 8356.

Claim 108

The monoclonal receptor tht reacts with glucitollysine and is produced and secreted by the hybridoma of claim 81.

Claim 109

A hybridoma having ATCC accession number HB 8357.

Claim 110

A hybridoma having ATCC accession number HB 8357.

Claim 111

The monoclonal receptor that reacts with glucitollysine and is produced and secreted by the hybridoma of claim 83.

Claim 112

A hybridoma having ATCC accession number HB 8358.

Claim 113

A hybridoma having ATCC accession number HB 8358.

Claim 114

The monoclonal receptor that reacts with glucitollysine and is produced and secreted by the hybridoma of claim 85.

Claim 115

A hybridoma having ATCC accession number HB 8359.

Claim 116

A hybridoma having ATCC accession number HB 8359.

Claim 117

The monoclonal receptor that reacts with glucitollysine and is produced and secreted by the hybridoma of claim 87.

Claim 118

A hybridoma having ATCC accession number HB 8354.

Claim 119

A hybridoma having ATCC accession number HB 8354.

Claim 120

The monoclonal receptor that reacts with glucitollysine and is produced and secreted by the hybridoma of claim 89.