Immunological assay for plasmin-digested products of stabilized fibrin
View Patent ↗A method of immunologically analyzing plasmin-digested products of stabilized fibrin, characterized by using a combination of a monoclonal antibody (a) which does not react with stabilized fibrin, fibrinogen, and plasmin-digested products of fibrinogen, but reacts with a neoantigen which is newly exposed in a D domain by digesting stabilized fibrin with plasmin, and a monoclonal antibody (b) which recognizes a site different from that recognized by the monoclonal antibody (a), and specifically reacts with plasmin-digested products of stabilized fibrin, wherein one of the monoclonal antibodies (a) and (b) is carried on a magnetic particle, and the other is labeled with an enzyme, and a chemiluminescent substrate is used as a substrate for the enzyme, is disclosed.
1. A method of immunologically analyzing plasmin-digested products of stabilized fibrin, the method comprising:
(a) bringing a sample suspected of containing plasmin-digested products of stabilized fibrin into contact with a first monoclonal antibody and a second monoclonal antibody,
wherein the first monoclonal antibody does not form a sandwich immunocomplex with stabilized fibrin, fibrinogen, or plasmin-digested products of fibrinogen, but reacts with a neoantigen in a D domain of stabilized fibrin which is newly exposed by digesting stabilized fibrin with plasmin, and the second monoclonal antibody does not form a sandwich immunocomplex with stabilized fibrin, fibrinogen, or plasmin-digested products of fibrinogen, but reacts with plasmin-digested products of stabilized fibrin and a neoantigen that is newly exposed in an E domain by digesting stabilized fibrin with plasmin,
wherein one of said first or second monoclonal antibodies is carried on a magnetic particle, and the other is labeled with an enzyme label;
(b) separating enzyme-labeled monoclonal antibody which forms an immunocomplex via plasmin-digested products of stabilized fibrin with the monoclonal antibody carried on a magnetic particle from enzyme-labeled monoclonal antibody which does not form the immunocomplex;
(c) adding a chemiluminescent substrate to the separated immunocomplex to generate a chemiluminescent signal; and
(d) analyzing the generated chemiluminescent signal, wherein the amount of chemiluminescent signal correlates with the amount of plasmin-digested products of stabilized fibrin in the sample.
2. The method according to claim 1 , wherein the chemiluminescent substrate is 1,2-dioxetane.
3. The method according to claim 1 , wherein the enzyme label is alkaline phosphatase.
4. A kit for immunologically analyzing plasmin-digested products of stabilized fibrin, said kit comprising
(a) a first monoclonal antibody which does not form a sandwich immunocomplex with stabilized fibrin, fibrinogen, or plasmin-digested products of fibrinogen, but binds to a neoantigen which is newly exposed in a D domain of stabilized fibrin by digesting stabilized fibrin with plasmin,
(b) a second monoclonal antibody which does not form a sandwich immunocomplex with stabilized fibrin, fibrinogen, or plasmin-digested products of fibrinogen, but binds to plasmin-digested products of stabilized fibrin and a neoantigen that is newly exposed in an E domain of stabilized fibrin by digesting stabilized fibrin with plasmin, and
(c) a chemiluminescent substrate,
wherein one of the first or second monoclonal antibodies is carried on a magnetic particle, and the other is labeled with an enzyme label, a substrate of which is the chemiluminescent substrate.
5. A method of immunologically analyzing plasmin-digested products of stabilized fibrin, the method comprising:
(a) bringing a sample suspected of containing plasmin-digested products of stabilized fibrin into contact with
(i) a first monoclonal antibody that does not form a sandwich immunocomplex with stabilized fibrin, fibrinogen, or plasmin-digested products of fibrinogen, but binds to a first epitope on a neoantigen in a D domain of stabilized fibrin which is newly exposed by digesting stabilized fibrin with plasmin, and
(ii) a second monoclonal antibody which does not form a sandwich immunocomplex with stabilized fibrin, fibrinogen, or plasmin-digested products of fibrinogen, but binds to plasmin-digested products of stabilized fibrin and a neoantigen that is newly exposed in an E domain of stabilized fibrin by digesting stabilized fibrin with plasmin, to form an immunocomplex of first monoclonal antibody-plasmin-digested products of stabilized fibrin-second monoclonal antibody, wherein said second monoclonal antibody carries a detectable label;
(b) separating unbound second monoclonal antibody from said immunocomplex;
(c) analyzing the amount of label in said sample, wherein the amount of label detected indicates the amount of plasmin-digested products of stabilized fibrin in said sample.
6. The method of claim 5 , wherein said first monoclonal antibody is carried on a magnetic particle.
7. The method of claim 5 , wherein said detectable label is an enzyme label.
8. The method of claim 7 further comprising the step of adding enzyme-specific chemiluminescent substrate to said immunocomplex.