Immunoassay reagents and methods of use thereof
First Claim
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1. A composition comprising at least two antibodies and a buffer selected from the group consisting of:
- a. 1.5 to 3.0 g/l sodium phosphate monobasic, monohydrate;
0.5 to 0.6 g/l potassium phosphate dibasic, trihydrate;
0.5 to 1.5 ml/l polyoxyethylenesorbitan monolaurate, 50 to 100 ml/l of 5% bovine serum albumin, 0.5 to 1.5 g/l sodium azide, 0.005 g/l, and water and which is at a pH of from about 5.5 to about 6.5;
b. 40 to 60 ml/l 10×
PBS, 0.75 to 1.25 g/l sodium azide, 0.1 to 0.3 ml/l polyoxyethylenesorbitan monolaurate, 45 to 65 g/l bovine serum albumin, and water and which is at a pH of about 7.0 to about 6.5;
c. 3.0 to 4.0 g/l Tris-hydrochloride, 0.75 to 1.2 g/l sodium azide, 7.5 to 12.5 g/l bovine serum albumin, 0.2 to 0.3 ml/l 25% hydrochloric acid, and water and which is at a pH of about 5.7 to about 6.5;
d. one part of a first solution comprising 75 to 125 ml/l 10 ×
PBS, 2.5 ml/l preservative, 0.25 to 0.75 ml/l of 50% polyoxyethylenesorbitan monolaurate, 2.5 to 7 g/l purified casein, 2.0 to 3.0 ml/l purified Type A gelatin, and water, and a second solution comprising 2.0 to 3.0 g/l sodium phosphate monobasic, monohydrate;
0.5 to 0.6 g/l potassium phosphate dibasic, trihydrate;
0.75 to 1.25 ml/l polyoxyethylenesorbitan monolaurate, 75 to 125 ml/l of 2.5 to 7.5% bovine serum albumin, 0.7 to 1.1 g/l sodium azide, and water, and which is at a pH of about 5.75 to about 6.25; and
e. 1.5 to 3.0 g/l sodium phosphate monobasic, monohydrate;
0.5 to 0.6 g/l potassium phosphate dibasic, trihydrate;
0.5 to 1.5 ml/l polyoxyethylenesorbitan monolaurate, 50 to 100 ml/l of 5% bovine serum albumin, 0.5 to 1.5 g/l sodium azide, 0.005 g/l, 40 to 60% glycerol, and water, and which is at a pH is about 5.5 to about 6.5.
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Abstract
The present invention provide reagents and methods of using the reagents, for example, on automated staining devices, that facilitate detection of two or more antigens in a sample simply and efficiently.
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Citations
92 Claims
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1. A composition comprising at least two antibodies and a buffer selected from the group consisting of:
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a. 1.5 to 3.0 g/l sodium phosphate monobasic, monohydrate;
0.5 to 0.6 g/l potassium phosphate dibasic, trihydrate;
0.5 to 1.5 ml/l polyoxyethylenesorbitan monolaurate, 50 to 100 ml/l of 5% bovine serum albumin, 0.5 to 1.5 g/l sodium azide, 0.005 g/l, and water and which is at a pH of from about 5.5 to about 6.5;
b. 40 to 60 ml/l 10×
PBS, 0.75 to 1.25 g/l sodium azide, 0.1 to 0.3 ml/l polyoxyethylenesorbitan monolaurate, 45 to 65 g/l bovine serum albumin, and water and which is at a pH of about 7.0 to about 6.5;
c. 3.0 to 4.0 g/l Tris-hydrochloride, 0.75 to 1.2 g/l sodium azide, 7.5 to 12.5 g/l bovine serum albumin, 0.2 to 0.3 ml/l 25% hydrochloric acid, and water and which is at a pH of about 5.7 to about 6.5;
d. one part of a first solution comprising 75 to 125 ml/l 10 ×
PBS, 2.5 ml/l preservative, 0.25 to 0.75 ml/l of 50% polyoxyethylenesorbitan monolaurate, 2.5 to 7 g/l purified casein, 2.0 to 3.0 ml/l purified Type A gelatin, and water, and a second solution comprising 2.0 to 3.0 g/l sodium phosphate monobasic, monohydrate;
0.5 to 0.6 g/l potassium phosphate dibasic, trihydrate;
0.75 to 1.25 ml/l polyoxyethylenesorbitan monolaurate, 75 to 125 ml/l of 2.5 to 7.5% bovine serum albumin, 0.7 to 1.1 g/l sodium azide, and water, and which is at a pH of about 5.75 to about 6.25; and
e. 1.5 to 3.0 g/l sodium phosphate monobasic, monohydrate;
0.5 to 0.6 g/l potassium phosphate dibasic, trihydrate;
0.5 to 1.5 ml/l polyoxyethylenesorbitan monolaurate, 50 to 100 ml/l of 5% bovine serum albumin, 0.5 to 1.5 g/l sodium azide, 0.005 g/l, 40 to 60% glycerol, and water, and which is at a pH is about 5.5 to about 6.5. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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- 40. A composition comprising at least two antibodies wherein at least one antibody is coupled to a poly (horseradish peroxidase) and at least a second antibody is coupled to a poly(alkaline phosphatase) and a buffer comprising 0.8 to 1.2 M Tris-hydrochloride, pH from 7.3 to 7.9, 0.025 to 0.075% polyoxyethylenesorbitan monolaurate and 2.5 to 3.5% goat serum.
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52. The method of claim 52, wherein the rabbit antibody is a rabbit monoclonal antibody.
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61. A method of detecting at least two antigens in a sample, comprising contacting a sample with a first antibody;
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denaturing the sample with a composition comprising 1 part of a first solution which comprises 1.1 to 1.3% hydrochloric acid, 0.020 to 0.030% preservative, and water; and
3 parts of a second solution comprising 0.1 to 0.3% polyoxyethylenesorbitan monolaurate, about 0.2 to 0.3% preservative, and water;
contacting the sample with a second antibody; and
visualizing the formation of at least two antigen-antibody complexes on the sample. - View Dependent Claims (62, 63, 64, 65, 66, 67, 68, 69, 70)
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71. A method of detecting at least two antigens in a sample, comprising contacting the sample with a composition comprising at least one first primary antibody and at least one second primary antibody;
- and detecting the formation of at least two antigen-antibody complexes on the sample, wherein the at least one first and second antibodies specifically bind to two antigens selected from the group consisting of;
CD3 and Caspase-3;
CD20 and CD3;
CD31 and Ki-67;
CD34 and Factor XIII subunit a;
CDX2 and CK7;
Ki-67 and Caspase-3;
M30 and Ki-67;
LCA and S-100;
CD20 and Ki-67;
Tyrosinase and S100;
Tyrosinase and MART-1;
Tyrosinase and A103;
P63 and CK5;
P63 and P504S;
P63 and P504S;
CK5/6 and Calretinin;
Estrogen receptor and Ki-67;
CK5 and CK17;
CD10 and Prostate specific antigen;
CD10 and Hepatic specific antigen;
Chromogranin A and Synaptophysin;
HMW CK and LMW CK;
CD20 and Caspase-3;
CD3 and Ki-67;
PAX-5 and CD5;
CD4 and CD8;
Kappa light chain and lambda light chain. - View Dependent Claims (72, 73, 74, 75, 76, 77, 78, 79, 80)
- and detecting the formation of at least two antigen-antibody complexes on the sample, wherein the at least one first and second antibodies specifically bind to two antigens selected from the group consisting of;
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81. A method of detecting two or more antigens in a sample, comprising
contacting a sample, which has been previously contacted with a primary antibody cocktail comprising at least one first primary antibody and at least one second primary antibody, with a composition comprising at least one first secondary antibody and at least one second secondary antibody, wherein the at least one first secondary antibody is coupled to a poly (alkaline phosphatase) moiety and the at least one second secondary antibody is coupled to a poly (horseradish peroxidase) moiety, and wherein the composition comprises a buffer suitable to stabilize the first and second secondary antibodies; - and
detecting the formation of at least two antigen-antibody complexes on the sample. - View Dependent Claims (82, 83, 84, 85, 86)
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87. A method of detecting two or more antigens in a sample, comprising
contacting a sample with a primary antibody cocktail comprising at least one first primary antibody and at least one second primary antibody, and subsequently contacting the sample with a composition comprising at least one first secondary antibody and at least one second secondary antibody, wherein the at least one first secondary antibody is coupled to a poly (alkaline phosphatase) moiety and the at least one second secondary antibody is coupled to a poly (horseradish peroxidase) moiety, and wherein the composition comprises a buffer suitable to stabilize the first and second secondary antibodies; - and
detecting the formation of at least two antigen-antibody complexes on the sample. - View Dependent Claims (88, 89, 90, 91, 92)
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Specification