Methods and compositions for immuno-histochemical detection
First Claim
Patent Images
1. A method of detecting a biological marker in a sample comprising:
- a. contacting the sample with at least one first binding agent that specifically binds to the biological marker in the sample such that the at least one first binding agent binds to the sample and forms a first complex;
b. contacting the first complex of a) with at least one second binding agent that specifically binds to the at least one first binding agent such that a second complex is formed, wherein the at least one second binding agent is linked to at least one first polymer;
c. contacting the second complex of b) with at least one third binding agent that specifically binds to the at least one second binding agent, wherein the at least one third binding agent is linked to at least one second polymer;
wherein at least one detectable agent is linked to at least one ofi. the at least one third binding agent;
ii. the at least one second polymer;
oriii. both the at least one third binding agent and the at least one second polymer;
wherein the at least one third binding agent binds to the second complex of b) and forms a third complex; and
d. detecting the at least one detectable agent of the third complex of c), thereby detecting the biological marker in the sample.
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Abstract
The invention relates to methods and compositions of detecting a biological marker in a sample. In certain embodiments, the methods and compositions comprise a first binding agent that specifically binds to a biological marker in a sample; a second binding agent linked to a first polymer, wherein the second binding agent specifically binds to the first binding agent; a third binding agent linked to a second polymer, wherein the third binding agent specifically binds to the second binding agent; and a detectable substance. In certain embodiments the biological markers are detected immunologically.
12 Citations
49 Claims
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1. A method of detecting a biological marker in a sample comprising:
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a. contacting the sample with at least one first binding agent that specifically binds to the biological marker in the sample such that the at least one first binding agent binds to the sample and forms a first complex; b. contacting the first complex of a) with at least one second binding agent that specifically binds to the at least one first binding agent such that a second complex is formed, wherein the at least one second binding agent is linked to at least one first polymer; c. contacting the second complex of b) with at least one third binding agent that specifically binds to the at least one second binding agent, wherein the at least one third binding agent is linked to at least one second polymer; wherein at least one detectable agent is linked to at least one of i. the at least one third binding agent; ii. the at least one second polymer;
oriii. both the at least one third binding agent and the at least one second polymer; wherein the at least one third binding agent binds to the second complex of b) and forms a third complex; and d. detecting the at least one detectable agent of the third complex of c), thereby detecting the biological marker in the sample. - 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, 40, 41, 42, 43, 44)
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45. A method of detecting a biological marker in a sample comprising
a. contacting the sample with at least one primary mouse or rabbit antibody that specifically binds to the biological marker in the sample, such that the at least one primary antibody binds to the sample forming a first complex; -
b. contacting the first complex of a) with i. at least one first secondary goat antibody linked to at least one first polymer, the at least one first secondary goat antibody comprising an antigen binding domain that specifically binds to a mouse antibody; and ii. at least one second secondary goat antibody linked to at least one second polymer, the at least one second secondary goat antibody comprising an antigen binding domain that specifically binds to a rabbit antibody, such that at least one secondary antibody binds to the first complex of a) forming a second complex; c. contacting the second complex of b) with at least one tertiary rabbit antibody linked to at least one third polymer, the at least one tertiary rabbit antibody comprising an antigen binding domain that specifically reacts with a goat antibody; wherein at least one horseradish peroxidase molecule is linked to at least one of 1. the at least one tertiary antibody; 2. the at least one third polymer;
or3. both the at least one tertiary antibody and the at least one third polymer; wherein the at least one tertiary antibody binds to the second complex of b) and forms a third complex; and d. detecting the horseradish peroxidase of the third complex of c), thereby detecting the biological marker in the sample.
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46. A method of detecting a biological marker in a sample comprising
a. contacting the sample with at least one primary mouse antibody that specifically binds to the biological marker in the sample such that the at least one primary mouse antibody binds to the sample forming a first complex; -
b. contacting the first complex of a) with at least one rabbit secondary antibody linked to at least one first polymer, the at least one rabbit secondary antibody comprising an antigen binding domain that specifically reacts with a mouse antibody such that the at least one rabbit antibody specifically binds to the first complex of a) forming a second complex; c. contacting the second complex of b) with at least one tertiary goat antibody linked to at least one second polymer, the at least one tertiary goat antibody comprising an antigen binding domain that specifically binds to a rabbit antibody; wherein at least one horseradish peroxidase molecule is linked to at least one of 1. the at least one tertiary antibody; 2. the at least one second polymer;
or3. both the at least one tertiary antibody and the at least one second polymer wherein the at least one tertiary antibody binds to the second complex of b) and forms a third complex; and d. detecting the horseradish peroxidase of the third complex of c), thereby detecting the biological marker in the sample.
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47. A method of detecting a biological marker in a sample comprising
a. contacting the sample with at least one primary mouse antibody that specifically binds to the biological marker in the sample such that the at least one primary mouse antibody binds to the sample forming a first complex; -
b. contacting the first complex of a) with at least one secondary rabbit antibody linked to at least one first polymer, the at least one secondary rabbit antibody comprising an antigen binding domain which specifically reacts with a mouse antibody such that the at least one secondary antibody binds to the first complex of a) forming a second complex; c. contacting the second complex of b) with i. at least one first tertiary goat antibody linked to at least one second polymer, the at least one first tertiary goat antibody comprising an antigen binding domain that specifically binds to a rabbit antibody; and ii. at least one second tertiary goat antibody linked to at least one third polymer, the at least one second tertiary goat antibody comprising an antigen binding domain which specifically reacts with a mouse antibody; wherein at least one horseradish peroxidase molecule is linked to at least one of 1. any tertiary antibody; 2. the at least one second polymer; 3. the at least one third polymer; 4. both the at least one second polymer and the at least one third polymer;
or5. any tertiary antibody and both the at least one second polymer and the at least one third polymer; wherein the at least one first and second tertiary antibodies bind to the second complex of b and form a third complex; and d. detecting the horseradish peroxidase of the third complex of c), thereby detecting the biological marker in the sample. - View Dependent Claims (48)
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49. A method of detecting a biological marker in a sample comprising
a. contacting the sample with at least one primary mouse or rabbit antibody that specifically binds to the biological marker in the sample, such that the at least one primary antibody binds to the sample forming a first complex; -
b. contacting the first complex of a) with at least one second binding agent comprising i. at least one first secondary goat antibody linked to at least one first polymer, the at least one first secondary goat antibody comprising an antigen binding domain that specifically binds to a mouse antibody; ii. at least one second secondary goat antibody linked to at least one second polymer, the at least one second secondary goat antibody comprising an antigen binding domain that specifically binds to a rabbit antibody; and iii. at least one hapten molecule linked to at least one of 1. the first or second secondary goat antibodies; 2. the first or second polymers;
or3. the first or second secondary goat antibody and the first or second polymers; such that at least one secondary antibody binds to the first complex of a) forming a second complex; c. contacting the second complex of b) with at least one tertiary rabbit, mouse, rat, pig, or goat antibody, linked to at least one third polymer, the at least one tertiary antibody comprising an antigen binding domain that specifically binds to at least one hapten molecule of b); wherein at least one horseradish peroxidase molecule is linked to at least one of 1. the at least one tertiary antibody; 2. the at least one third polymer;
or3. both the at least one tertiary antibody and the at least one third polymer; wherein the at least one tertiary antibody binds to the second complex of b) and forms a third complex; and d. detecting the horseradish peroxidase of the third complex of c), thereby detecting the biological marker in the sample.
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Specification