US20180106793A1 - Reagent and method for assaying thrombin-antithrombin complex - Google Patents

Reagent and method for assaying thrombin-antithrombin complex Download PDF

Info

Publication number
US20180106793A1
US20180106793A1 US15/562,839 US201615562839A US2018106793A1 US 20180106793 A1 US20180106793 A1 US 20180106793A1 US 201615562839 A US201615562839 A US 201615562839A US 2018106793 A1 US2018106793 A1 US 2018106793A1
Authority
US
United States
Prior art keywords
tat
antibody
complex
antithrombin
reagent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US15/562,839
Other languages
English (en)
Inventor
Tatsuya Yoshida
Yuhang Yang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LSI Medience Corp
Original Assignee
LSI Medience Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2015074168A external-priority patent/JP6594641B2/ja
Priority claimed from JP2015074173A external-priority patent/JP6615474B2/ja
Application filed by LSI Medience Corp filed Critical LSI Medience Corp
Assigned to LSI MEDIENCE CORPORATION reassignment LSI MEDIENCE CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YANG, Yuhang, YOSHIDA, TATSUYA
Publication of US20180106793A1 publication Critical patent/US20180106793A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G01N2333/8121—Serpins
    • G01N2333/8128—Antithrombin III
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/974—Thrombin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/224—Haemostasis or coagulation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis

Definitions

  • the present invention relates to a reagent and a method for assaying the thrombin (T)-antithrombin (AT) complex (TAT) in a biological sample.
  • TAT thrombin-antithrombin complex
  • DIC disseminated intravascular coagulation
  • TAT complex is a protein complex generated in blood in the course of blood coagulation and the quantification of TAT complexes in blood is useful for the diagnosis of thrombosis, such as disseminated intravascular coagulation (DIC).
  • DIC disseminated intravascular coagulation
  • the abundance of TAT complex is approximately 1/100,000 of the abundance of free antithrombin and, therefore, the measurement of TAT complex is not easy.
  • Examples of a currently prevailing TAT quantification method include reagent kits employing enzyme immunoassay (ELISA) technologies, such as Enzygnost® TAT micro from Siemens AG, and reagent kits employing chemiluminescent enzyme immunoassay (CLEIA) technologies, such as STACIA® CLEIA TAT from LSI Rulece Co.
  • ELISA enzyme immunoassay
  • CLIA chemiluminescent enzyme immunoassay
  • Patent Documents 1-3 and 5 have reported reagent systems for the TAT assay based on latex agglutination assay, any of which aims to assay samples prepared by diluting TAT complexes synthesized ex vivo with a buffer solution.
  • any reagent that enables the precise concentration of TAT complexes in human samples to be measured using latex agglutination assay.
  • the effect of the cross-reactivity of an antibody used is circumvented by establishing a TAT assay reagent based on the specificity thereof or by adding an additive agent.
  • Patent Document 4 discloses an assay of TAT by a sandwich method using an antibody with no cross-reactivity but the assay lacks sufficient sensitivity for clinical use. Also, in any of the documents, it is not examined how much the pH during the measurement reaction influences on the measurement results when TAT complexes in samples are quantified. There has been no attempt so far to allow the reaction to proceed at an acidic pH in the TAT assay by latex immunoagglutination assay.
  • Patent Document 1 Japanese Unexamined Patent Publication No. 2001-289850
  • Patent Document 2 Japanese Unexamined Patent Publication No. Hei7-238099
  • Patent Document 3 Japanese Unexamined Patent Publication No. 2002-316999
  • Patent Document 4 Japanese Unexamined Patent Publication No. Hei3-48158
  • Patent Document 5 Japanese Unexamined Patent Publication No. 2001-228153
  • An object of the present invention is to provide a reagent and a method for assaying TAT complexes in a biological sample based on latex agglutination assay, which does not essentially require B/F separation and washing operations, and to provide a method for selecting an antibody for use in the assay.
  • the TAT assay has been considered to be clinically useful in respect of improving the sensitivity and the specificity of the diagnostic criteria for DIC or in respect of the availability of the measurement as a diagnosis by exclusion, in which DIC is seen to be negative where the measured TAT value is normal.
  • ELISA which needs laborious operations
  • CLEIA which requires a special machine, are currently prevailing TAT assay methods is likely a reason for the slow spread of the assay.
  • latex agglutination assay does not comprise the step of B/F separation by washing and the like, it is an objective in the establishment of a reagent and an assay method to overcome the cross-reactivity to substances other than the original target of interest, such as free antithrombin and the like.
  • TAT assay reagent with high sensitivity and high accuracy was successfully produced by using an antibody with cross-reactivity and utilizing the difference in cross-reactivity of the antibody. That is, the inventors found that TAT complexes in a biological sample were successfully measured accurately while minimizing the influence of free antithrombin by using an antibody, as one of the antibodies, selected by indirect inhibition ELISA and having a reactivity to TAT that is 100 or more times higher than the reactivity to free antithrombin.
  • TAT complexes in a biological sample were successfully measured sensitively and specifically by allowing the agglutination reaction to proceed under weak acidic conditions, and thereby completed the present invention.
  • a thrombin-antithrombin complex (TAT) assay reagent comprising: an antibody bound to a latex particle which binds to the antithrombin part of the TAT complex and recognizes the complex; and an antibody bound to a latex particle which binds to the thrombin part of the TAT complex and recognizes the complex: wherein the antibody binding to the antithrombin part of the TAT complex and recognizing the complex has a reactivity to TAT that is 100 or more times higher than the reactivity to free antithrombin.
  • the TAT assay reagent according to [1] wherein the assay reagent is designed to achieve a pH of 5.8 to 6.6 during the assay.
  • latex particles coupled to the antibody which binds to the antithrombin part of the TAT complex and recognizes the complex
  • latex particles coupled to the antibody which binds to the thrombin part of the TAT complex and recognizes the complex
  • a first reagent comprising a buffer solution at a pH of 5.8 to 6.6.
  • a method for screening an antibody for use in the TAT assay of a sample separated from a living body by latex agglutination assay comprising: preparing candidate antibodies: allowing the candidate antibodies to react with a certain amount of free antithrombin; subjecting the thus-obtained reaction liquids to an enzyme immunoassay using a TAT-immobilized substrate to quantify the reactivity of the antibodies to free antithrombin; comparing the reactivity of the antibodies to free antithrombin with that to TAT; and selecting an antibody having a reactivity to TAT that is 100 or more times higher than the reactivity to free antithrombin.
  • a thrombin-antithrombin complex (TAT) assay reagent comprising: a first antibody bound to a latex particle which binds to the antithrombin part of the TAT complex and recognizes the complex; and a second antibody bound to a latex particle which binds to the thrombin part of the TAT complex and recognizes the complex: wherein the assay reagent is designed to achieve a pH of 5.8 to 6.6 during the assay.
  • TAT assay reagent according to [6] comprising: a second reagent comprising
  • a method for measuring TAT complexes in a sample separated from a living body comprising measuring TAT by performing latex immunoagglutination reaction under a condition of pH 5.8 to 6.6 using the TAT assay reagent according to [6] or [7].
  • the above-described problems can be solved. That is, antibodies available for latex agglutination assay can be efficiently selected and, thus, a reagent excellent in fidelity, sensitivity and specificity can be timely marketed.
  • the reagent for latex agglutination assay provided in the present invention can accurately measure (quantify) a tiny amount of TAT in a biological sample while circumventing the effect of background substances such as plasma matrices. Also, it is an advantage of the reagent that the reagent allows the measurement on a general-purpose auto-analyzer and eliminates limitations, such as manual operation steps and measurement on a special machine.
  • a reagent providing both excellent sensitivity and specificity by keeping the pH during the reaction in the weak acidic range. It was found that the increase in pH tended to decrease in the saline blank value, the plasma blank value and the reactivity to TAT in a pH-dependent manner. It was a surprising phenomenon found by the inventors that the reactivity was decreased at a pH higher than neutral. Thus, it is now possible to provide a reagent having high reactivity and high specificity by keeping the pH during the reaction in the weak acidic range.
  • FIG. 1 shows a schematic diagram of the TAT assay method based on latex agglutination assay.
  • FIG. 2 shows a schematic diagram of the reaction system by indirect inhibition ELISA.
  • FIG. 3 shows the results from the evaluation of the reactivity of the clone TAT-5 to each indicated antigen in indirect inhibition ELISA.
  • FIG. 4 shows the results from the evaluation of the reactivity of a latex reagent coupled to each indicated antibody against TAT.
  • FIG. 5 shows the results from the evaluation of the cross-reactivity of a latex reagent coupled to each indicated antibody against free antithrombin.
  • FIG. 6 shows the change in baseline absorbance observed when the pH was changed from 6.0 to 7.2.
  • FIG. 7 shows the effect of the change in pH on the reactivity to TAT observed when the pH was changed from 6.0 to 7.2, where the plasma baseline has been subtracted.
  • FIG. 8 shows the change in baseline absorbance observed when the pH was changed from 5.7 to 6.2 (where the buffer solution for the first reagent is Bis-Tris buffer).
  • FIG. 9 shows the change in baseline absorbance observed when the pH was changed from 5.7 to 6.2 (where the buffer solution for the first reagent is MES buffer).
  • FIG. 10 shows the correlation between the results from the measurement on clinical samples evaluated by using the reagent of the present invention and using CLEIA.
  • TAT assay reagent which is composed of an antibody binding to the antithrombin part of the TAT complex and recognizing the complex as a first antibody and an antibody binding to the thrombin part of the TAT complex and recognizing the complex as a second antibody, but the scope of the present invention will not be limited thereto.
  • the TAT assay reagent of the present invention is a reagent for an immunoassay to measure TAT complexes in a biological sample by latex agglutination assay in a sandwich system using latex particles coupled to either of two different antibodies against TAT.
  • the first antibody is not limited as long as it is an anti-TAT antibody which binds to the antithrombin part of the TAT complex and recognizes the complex, but it is preferable to use an anti-TAT antibody having a low level of cross-reactivity to free antithrombin because the abundance of TAT complexes in blood is very low relative to that of free antithrombin.
  • the TAT assay reagent of the present invention is preferably a TAT assay reagent comprising a first antibody bound to a latex particle which binds to the antithrombin part of the TAT complex and recognizes the complex, and a second antibody bound to a latex particle which binds to the thrombin part of the TAT complex and recognizes the complex, wherein the first antibody has a reactivity to TAT that is 100 or more times higher than the reactivity to free antithrombin.
  • the two different antibodies may be coupled to two different types of particles, respectively, or the plural types of antibodies may be coupled to one type of particle, or either one of the antibodies may be coupled to several types of particles and then mixed for use.
  • the reactivity of the first antibody to TAT may be 100 or more times higher than the reactivity to free antithrombin and is preferably 1,000 or more times higher and more preferably 10,000 or more times higher than the reactivity to free antithrombin.
  • the maximum extent of cross-reactivity is not particularly specified because a lower level of cross-reactivity is better, but the cross-reactivity to free antithrombin may be, for example, 100,000 times or 50,000 times less than the reactivity to TAT.
  • either free antithrombin or TAT may be used for the immunization in animals other than human and the resulting antibody may be used in the present invention as long as it can bind to the antithrombin part of the TAT complex and recognize the complex.
  • the phrase “binding to the antithrombin part” means binding to antithrombin in a complex (TAT) formed by the association of free antithrombin, which is most abundant in a sample, with free thrombin. Accordingly, in cases where the antithrombin in a conformation achieved when it forms the complex is referred to as antithrombin in the complex-form structure and the antithrombin in a conformation achieved when it does not form the complex is referred to as antithrombin in the free-form structure (free antithrombin), the phrase “binding to the antithrombin part” means binding to the antithrombin in the complex-form structure.
  • the antithrombin in the free-form structure has a conformation different from that of the antithrombin in the complex-form structure. The reason is that the antithrombin in the free-form structure changes its conformation through the formation of a complex associated with free thrombin and maintains the changed conformation.
  • the second antibody is not limited as long as it is an antibody which can bind to the thrombin part of the TAT complex and recognize the complex, but an antibody which specifically reacts with thrombin can be used. Even an antibody which has cross-reactivity to free thrombin may often be used because free thrombin molecules are quite rare in a sample. Those skilled in the art will be able to select and use appropriate antibodies.
  • either free thrombin or TAT may be used for the immunization in animals other than human and the resulting antibody may be used in the present invention as long as it can bind to the thrombin part of the TAT complex and recognize the complex.
  • the phrase “binding to the thrombin part” means binding to thrombin in a complex (TAT) formed by the association of free thrombin, which is present in a sample, with antithrombin. Accordingly, in cases where the thrombin in a conformation achieved when it forms the complex is referred to as thrombin in the complex-form structure and the thrombin in a conformation achieved when it does not form the complex is referred to as thrombin in the free-form structure, the phrase “binding to the thrombin part” means binding to the thrombin in the complex-form structure.
  • the thrombin in the free-form structure has a conformation different from that of the thrombin in the complex-form structure. The reason is that the thrombin in the free-form structure changes its conformation through the formation of a complex associated with antithrombin and maintains the changed conformation.
  • Either polyclonal or monoclonal antibodies may be used for the above-described first and second antibodies. Those skilled in the art will be able to obtain these antibodies according to known procedures.
  • Animals such as sheep, horse, goat, rabbit, mouse, rat, and the like may be used as animals to be immunized with an immunogen for the preparation of an antibody, and rabbit, goat, and the like are preferably used especially for the preparation of polyclonal antibodies.
  • monoclonal antibodies can be obtained by known methods to prepare hybridoma cells, and mouse, rat, rabbit or the like is preferably used in that case.
  • TAT may be used as an immunogen, as described above.
  • an antibody produced by using a complex associated with vitronectin, VTAT, as an immunogen can also be used in the present invention.
  • antithrombin and thrombin may be used for the first and second antibodies, respectively.
  • TAT complexes purified from a raw material that is, a sample collected from a living body or TAT complexes synthesized in vitro by combining free thrombin and free antithrombin molecules may be used.
  • the synthetic TAT complexes may be, for example, TAT complexes obtained by incubating in vitro thrombin and antithrombin molecules available as biologics, while TAT complexes expressed using a known translation system such as those in E. coli , mammalian cells, insect cells infected with baculovirus, and the like may be recovered, purified and used as an immunogen.
  • partial peptides of antithrombin and thrombin may be used for the production of the first and second antibodies, respectively.
  • a method of selecting a peptide sequence for an antigen a method of synthesizing a peptide fragment, and an immunization method, known methods can be used.
  • the assay method based on latex agglutination assay will be described by reference to FIG. 1 .
  • FIG. 1 when the first antibody binds to the antithrombin part of the TAT complex and the second antibody binds to the thrombin part of the TAT complex, agglutination of the latex particles occurs and allows the determination of the TAT concentration based on the absorbance measured at that time.
  • the ratio of TAT to free antithrombin which fails to form TAT both present in a living body is considered to range from 1:60,000 to 1:110,000 on the basis of the range measured in normal subjects and generally considered to be approximately 1:100,000. Moreover, although it is known that the ratio may be changed in patients with sepsis and/or hepatic diseases, the ratio is considered to be around 1:50,000 even in cases where the amount of free antithrombin is relatively low. Thus, if the first antibody also has reactivity to free antithrombin, the quantification of TAT will become difficult. Then, an antibody having a low level of reactivity to free antithrombin should be used for the quantification of TAT.
  • the reactivity of the antibody to TAT should be 50,000 to 100.000 or more times higher than the reactivity to free antithrombin on the calculation basis, it was found in the present invention as demonstrated in the Examples below that the quantification of TAT is fully possible by using an antibody as the first antibody where the antibody has a reactivity to TAT that is 100 or more times higher than the reactivity to free antithrombin.
  • the phrase “a reactivity to TAT that is 100 or more times higher than the reactivity to free antithrombin” refers to a case where the ratio of the affinities for individual antigens (TAT and free antithrombin) is 100 or more, a case where the ratio of the amounts of antigens required to exhibit a certain inhibition rate in measurement by means of indirect inhibition ELISA described below is 100 or more, and the like.
  • an antibody binding to the antithrombin part of the TAT complex and recognizing the complex (a candidate antibody for the first antibody) is prepared.
  • Such an antibody as an antibody directed against a binding site on the antithrombin part of the TAT complex and recognizing the complex may be selected from antibodies obtained in advance by a method, such as the method described below to prepare monoclonal antibodies by hybridoma cells.
  • a previously known antibody directed against a binding site on the antithrombin part of the TAT complex and recognizing the complex would likewise be used in the assessment system below.
  • a candidate antibody is allowed to react with a solution containing TAT or an antigen capable of inhibiting the reaction with TAT in a certain amount (for example, 0.1, 0.5, 1, 5, 10, 50 ⁇ g/mL) for a sufficient period of time (for example, 12 hours). Then, the reaction liquid is allowed to react with a TAT-immobilized substrate for a certain period of time. Subsequently, after washing, a labeled secondary antibody is used to measure the amount of the antibody bound to the TAT on the substrate (the percentage of residual antibody).
  • a certain amount of TAT complexes are immobilized onto a substrate, such as a plate, under a condition where any antigen that inhibits the reaction with the corresponding antibody is not present.
  • a substrate such as a plate
  • TAT antigen
  • the above-described candidate antibody for the first antibody in each concentration (for example, 0.04 to 1 ⁇ g/mL) is allowed to react with the above-described TAT-immobilized substrate for a certain period of time under a condition where any antigen that inhibits the reaction with the corresponding antibody is not present.
  • a labeled secondary antibody (anti-mouse IgG-HRP) is used to measure the amount of the antibody bound to the TAT on the substrate.
  • concentration of the antibody which gives an absorbance of around 1.0 (corresponding to 1000 according to the method described in Table 1) is determined.
  • This antibody concentration can be considered to be a concentration of the antibody in the inhibition with the antigen (Table 3; concentration during the reaction [ ⁇ g/mL]).
  • the candidate antibody in a concentration determined by the above-described method is allowed to react with a solution containing TAT or free antithrombin in a certain amount (for example, 0.1, 0.5, 1, 5, 10, 50 ⁇ g/mL) for a sufficient period of time (for example, 12 hours). Then, the reaction liquid is allowed to react with a TAT-immobilized substrate for a certain period of time. Subsequently, after washing, a labeled secondary antibody (anti-mouse IgG-HRP) is used to measure the amount of the antibody bound to the TAT on the substrate (the percentage of residual antibody).
  • a labeled secondary antibody anti-mouse IgG-HRP
  • the percentage of residual antibody can be calculated relative to the detection value obtained for the case where absorption with antigen is not performed, which is considered as 100%.
  • the antibody capable of binding to TAT is decreased in amount and thus the antibody detected by the labeled secondary antibody is decreased in amount (the percentage of residual antibody is decreased), while in cases where the reactivity of the antibody to free antithrombin is low, a more amount of the antibody remains capable of binding to TAT and thus the antibody detected by the labeled secondary antibody is increased in amount (the percentage of residual antibody is increased).
  • This percentage of residual antibody will be compared with a percentage of residual antibody obtained when the antibody is first reacted with TAT (the inhibition reaction is performed with TAT) and then the reaction liquid is reacted with the immobilized TAT.
  • the amount of TAT required to achieve the same percentage of residual antibody is calculated based on the above-described result from the inhibition with TAT.
  • the amount of the TAT inhibitory antigen required to achieve a percentage of residual antibody of 50% is less than 0.50 ⁇ g/mL when the inhibition is performed with TAT, the reactivity to TAT can be considered to be 100 or more times higher than the reactivity to free antithrombin.
  • the thus selected antibody can be selected as a first antibody.
  • its affinity (Kd) for TAT is preferably equal to or less than 10 ⁇ 8 .
  • Kd affinity for TAT
  • Examples of the antibody used in the present invention include antibody fragments.
  • the antibody fragments are fragments of a desired antibody, which moreover have the same reactivity as that of the original antibody.
  • Examples of an antibody fragment that can be used in the present invention include fragments such as Fab, Fab′, F(ab′) 2 , or Fv. Any of these fragments can be obtained, for example, by digestion of an antibody with a protein degradation enzyme according to a conventional method, followed by separation and purification according to conventional methods for protein separation and purification. These fragments may be directly immobilized onto latex particles and used, while fragments may be prepared as Fab′ or F(ab′) 2 fragments and immobilized onto latex particles. Fab′ and F(ab′) 2 fragments are more preferable in consideration of avoiding a non-specific reaction of an antibody to Fc fragments.
  • An antibody used in the present invention can be obtained by first producing anti-TAT antibodies (candidate antibodies) by a production method for monoclonal antibodies with hybridoma cells, and the like, and then selecting an antibody which shows a low level of cross-reactivity to free antithrombin, specifically an antibody having a reactivity to TAT that is 100 or more times higher than the reactivity to free antithrombin, from the anti-TAT antibodies (candidate antibodies) according to the procedures and criteria as described above.
  • the candidate antibodies can be obtained, for example, by a production method for monoclonal antibodies with hybridoma cells produced by a known cell fusion method.
  • Antibody-producing cells may be selected from animals except for human, for example, mouse, rat, guinea pig, and the like.
  • Hybridoma cells and monoclonal antibodies can be prepared according to conventional methods, for example, methods described in “Zoku-Seikagaku Jikken Koza” (Biochemical Experiment Training Course; The Japanese Biochemical Society, ed.) or “Men-eki Seikagaku Kenkyu-hou” (Immuno-biochemical research methods; The Japanese Biochemical Society, ed.).
  • the second antibody is not particularly limited as long as it is an antibody which binds to the thrombin part of the TAT complex and recognizes the complex.
  • its affinity (Kd) for TAT is preferably equal to or less than 10 ⁇ 8 .
  • Kd affinity for TAT
  • those skilled in the art will be able to appropriately select an antibody suitable for a latex reagent based on the affinity value for TAT.
  • antibodies produced similarly to the first antibody can be used.
  • the combination of the first antibody and the second antibody is not particularly limited as long as it allows the assay of TAT by latex agglutination assay, but a combination of the antibodies which is minimally affected by the matrices contained in a biological sample such as plasma (background) is preferably selected.
  • the reagent of the present invention is preferably a reagent capable of quantifying TAT complexes at a concentration of 10 to 15 ng/mL, more preferably a reagent capable of quantifying TAT complexes at a concentration of 3 to 4 ng/mL, and further preferably a reagent capable of quantifying TAT complexes even at a concentration of around 1 ng/mL, in a biological sample.
  • the latex particles to which the above-described first and second antibodies are coupled are not particularly limited as long as they can be used in the latex agglutination reaction, but they have an average particle size of preferably 0.05 ⁇ m to 0.5 ⁇ m and more preferably 0.2 to 0.4 ⁇ m.
  • latex particles only one type of latex particle or multiple types of latex particles may be used.
  • a combination of latex particles with different particle sizes may be used. Because it is practically difficult to manufacture latex particles with a single particle size, any latex particle is specified with the average particle size of all particles. Accordingly, in the reference to an average particle size of 0.05 ⁇ m to 0.5 ⁇ m, a case comprising latex particles outside this range may also be included in the present invention.
  • latex particles with different particle sizes are included in a given latex particle is within the common sense of those skilled in the art and those skilled in the art will be able to establish a latex reagent by using a solution containing a group of particles having a not highly heterogeneous size distribution.
  • the average particle size can be measured according to a known method and, for example, can be calculated on the basis of image analysis using a transmission electron microscope system.
  • the latex particle according to the present invention is not particularly limited as long as it is commonly used in the art, but examples of the latex particle include particles made from homopolymers (for example, polystyrene, methacrylate polymers, acrylate polymers, and the like) composed of polymerized vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylate, methacrylate, and the like; particles made from butadiene copolymers (for example, styrene-butadiene copolymer, methylmethacrylate-butadiene copolymer, acrylonitrile-butadiene copolymer, and the like); and particles made from other copolymers (for example, styrene-styrenesulfonate copolymer, methacrylate copolymers, acrylate copolymers, vinyl chloride-acrylate copolymers, and the like).
  • homopolymers for example, poly
  • the latex particle examples include particles carrying a carboxyl group, a primary amino group, a carbamoyl group (—CONH 2 ), a hydroxyl group, an aldehyde group, or the like as a functional group and having a base body composed of any of the above-described organic particulates.
  • the antibodies may be immobilized according to a known method and can be immobilized by treatments commonly used in the art, such as, for example, suspending the antibody and latex particles in a buffer solution, allowing them to react at 25° C. for one hour, followed by centrifugation, blocking treatment, and the like.
  • a method to immobilize the antibodies onto latex particles through chemical linkage or through biotin-avidin interaction may be selected.
  • the coupling of the antibodies to latex particles is performed under conditions where the antibodies can maintain the above-described reactivity and specificity to TAT.
  • an antibody-immobilized latex liquid may be prepared for every antibody as a first latex particle and a second latex particle onto which the first antibody and the second antibody have been immobilized, respectively, while the reagents may be prepared by immobilizing the first antibody and the second antibody onto a single type of latex particles.
  • reagents may be prepared by immobilizing the first antibody and the second antibody onto a single type of latex particles.
  • the reagent of the present invention may be a single-reagent system or a two-reagent system. If the reagent of the present invention is a single-reagent system. TAT complexes in a biological sample can be measured by adding a suspension of latex particles carrying the immobilized antibodies to the biological sample, and causing an antigen-antibody reaction. If the reagent of the present invention is a two-reagent system. TAT complexes in a biological sample can be measured by adding the first reagent mainly composed of buffer ingredients to the biological sample and then further adding the second reagent containing latex particles onto which the antibodies have been immobilized, and causing an antigen-antibody reaction.
  • the degree of agglutination of latex particles can be measured, for example, using absorbance, while the concentration of TAT complexes in a sample can be quantified by searching for a concentration corresponding to the degree of agglutination on a previously obtained standard curve of the reference. Additionally, the measurement of absorbance may be performed at a measurement wavelength of normally 340 nm to 1000 nm and preferably 500 nm to 900 nm.
  • the kinetics of agglutination or the variation in agglutination over a fixed time interval during the proceeding of the latex agglutination reaction can be determined by photometry.
  • the kinetics of absorbance changes or the variation in absorbance over a fixed time interval within the period from 30 seconds to 5 minutes after the start of the latex agglutination reaction can be determined by photometry.
  • the reaction temperature is preferably from 10 to 50° C. and more preferably from 20 to 40° C.
  • the reaction time can be appropriately determined and the measurement may be performed, for example, within a reaction time of 10 to 15 minutes on a general-purpose auto-analyzer.
  • reaction temperature the reaction time
  • the measurement wavelength the measurement time
  • the reagent composition the latex concentration
  • concentration of an antibody to be immobilized onto latex particles the concentrations of various additive agents in analysis using an optical instrument or a general-purpose auto-analyzer.
  • the concentration of latex particles used in the present invention is not particularly limited as long as it is a concentration applicable to a reagent for an immunological assay based on latex agglutination assay, but the concentration of latex particles during the reaction required for the TAT assay is preferably 0.005% (w/v) to 0.2% (w/v) and more preferably 0.01% (w/v) to 0.1% (w/v).
  • the test sample applicable to the reagent of the present invention is not particularly limited as long as it is a test sample potentially containing TAT complexes, but it is preferably a biological sample.
  • the biological sample may include cultured cells but also those preferably used in the measurement with serum or plasma.
  • the biological sample is preferably a sample derived from any mammalian animal and more preferably a sample derived from human.
  • the reagent of the present invention may further comprise, in addition to the latex particles onto which the antibodies have been immobilized, excipients which can be added to a reagent for an immunological assay based on latex agglutination assay, such as, for example, a buffer, an agglutination promoter, a non-specific binding suppressor, a sensitizing agent, and the like.
  • excipients which can be added to the reagent of the present invention include sodium alginate, propylene glycol alginate, and the like.
  • a water soluble polymer or protein is preferably used as an agglutination promoter which can be added to the reagent of the present invention.
  • agglutination promoter examples include water soluble polymers such as dextran and dextran sulfate, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, and the like, albumins such as bovine serum albumin; and globulins such as ⁇ -globulin.
  • a third antibody may be added for use.
  • an antibody having a different rate of reaction is preferably used as the third antibody in cases where, for example, the measurable range of the TAT assay reagent is desired to cover a broad range from a low concentration to a high concentration.
  • the buffer solution has a buffer capacity at a pH of preferably 5.8 to 6.6, more preferably 6.0 to 6.4, further preferably 6.1 to 6.3, particularly preferably 6.15 to 6.25 and most preferably about 6.2.
  • the pH of the reagent should be adjusted to 5.8 to 6.6, while in the case of a two-reagent system the reagents should be composed such that the pH reaches 5.8 to 6.6 when they are mixed.
  • the reagent system comprises a first reagent mainly composed of buffer ingredients and a second reagent containing latex particles onto which the antibodies have been immobilized
  • the pH of the first reagent is adjusted to 5.8 to 6.6 and the pH of the mixture reaches 5.8 to 6.6 when both the reagents are mixed.
  • the pH may be adjusted by a pH modifying agent and is preferably adjusted by a buffer solution.
  • a buffer solution such as Tris buffer, Bis-Tris buffer, phosphate buffer, or Good's buffer is preferably used and the concentration of the buffer solution during the reaction is preferably 10 to 500 mmol/L and more preferably 20 to 200 mmol/L.
  • the pH may be adjusted additionally by a pH modifying agent and the like.
  • buffer solutions such as Tris buffer, phosphate buffer, glycine buffer, borate buffer, citrate buffer, acetate buffer, or Good's buffer
  • chelating agents such as EDTA, CyDTA, DTPA.
  • EGTA, NTA, and NTP salts, such as sodium chloride, potassium chloride, sodium sulfate, calcium sulfate, magnesium sulfate, calcium carbonate, and sodium carbonate
  • non-ionic surfactants such as fatty acid diethanolamides, polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, fatty acid sorbitan esters, alkyl polygulcosides, alkyl monoglyceryl ethers, polyoxyethylene sorbitan fatty acid esters, fatty acid alkanolamides, and alkyl glycosides.
  • the reagent of the present invention may include TAT complexes which can be used as a standard substance.
  • the TAT complexes may be TAT complexes purified from a living body or synthesized by recombinant DNA technology and the like.
  • the synthetic TAT complexes can be obtained, for example, by incubating in vitro thrombin and antithrombin molecules available as biologics.
  • TAT complexes can also be synthesized by recovering and purifying and mixing the components which have been expressed using a known translation system in E. coli , mammalian cells, insect cells infected with baculovirus, and the like.
  • a commercially available human thrombin formulation (manufactured by Japan Blood Products Organization) and a commercially available antithrombin formulation (manufactured by Japan Blood Products Organization) were separately diluted with PBS (produced by dissolving Dulbecco's PBS ( ⁇ ) powder “Nissui (manufactured by Nissui Pharmaceutical Co., Ltd.)” to a concentration of 9.6 g/L) and those dilutions were mixed at a molar ratio of 1:3 and then allowed to react at 37° C. for 30 minutes. After the 30 minutes, DFP (diisopropyl fluorophosphate, manufactured by Wako Pure Chemical Industries, Ltd.) was added to a concentration of 0.75 mM to stop the reaction.
  • PBS produced by dissolving Dulbecco's PBS ( ⁇ ) powder “Nissui (manufactured by Nissui Pharmaceutical Co., Ltd.)” to a concentration of 9.6 g/L
  • TAT fractions were recovered after identification by SDS-PAGE.
  • the obtained TAT fraction was diluted with saline containing 0.5% BSA and analyzed using a CLEIA reagent (STACIA® CLEIA TAT manufactured by LSI Rulece Co.) to determine the concentration.
  • STACIA® CLEIA TAT manufactured by LSI Rulece Co. a CLEIA reagent
  • a cell fusion method was carried out according to the method described in Tamie Ando and Tatsuo Iwasaki, “Monoclonal Antibody/Hybridoma and ELISA” (Kodansha Ltd.).
  • the synthetic TAT complex prepared in Example 1 in an amount of 50 ⁇ g was mixed with Freund's complete adjuvant (manufactured by DIFCO) to provide an administered antigen.
  • the antigen was administered to BALB/c mice (female, four weeks old) three times at an interval of two weeks and 25 ⁇ g of the antigen, that is, half the amount of the original administered antigen was injected intravenously at the fourth administration.
  • lymphocytes were isolated from the spleen and mixed with P3x63-Ag.8 myeloma cells and then fused using polyethylene glycol (PEG 4000, manufactured by Merck).
  • PEG 4000 polyethylene glycol
  • Hybridoma cells were selected in HAT selection medium and then screened one week later for hybridoma clones producing an antibody of interest on the basis of the binding activity for the synthetic TAT complex. That is, the synthetic TAT complex was individually diluted with 0.05 M carbonate buffer (pH 9.5) to a concentration of 0.2 ⁇ g/mL and added at 50 ⁇ L/well to an immuno plate (Maxisorp, manufactured by NUNC). After the reaction at 4° C. overnight, each well was washed three times with PBS containing 0.05% Tween-20 and then blocked by adding thereto 100 ⁇ L of PBS containing 1.0% BSA.
  • the culture supernatant was added in a volume of 50 ⁇ L to each well and allowed to react at 37° C. for one hour and then each well was washed three times with PBS containing 0.05% Tween-20.
  • a peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by Dako) was diluted 1000 times in PBS containing 0.05% Tween-20 and then added in a volume of 50 ⁇ L to each well.
  • the absorbance was measured at 492 nm on a plate spectrophotometer (EL312e, manufactured by BioTek Instruments, Inc.). Cells producing antibodies showing good reactivity to the synthetic TAT complex were selected and then cloned by limiting dilution. Ten days later, those cells were further screened to obtain hybridoma clones producing antibodies which react with the synthetic TAT complex.
  • EL312e plate spectrophotometer
  • Anti-thrombin antibodies were obtained by a method similar to that in Example 2 using thrombin as an immunizing antigen. Antibodies specifically reacting with thrombin were selected and one of those clones was used as an anti-thrombin antibody (T-1).
  • the reactivity of each antibody was evaluated by indirect inhibition ELISA.
  • a schematic model of the reaction system by indirect inhibition ELISA is shown in FIG. 2 .
  • TAT-recognizing antibody (anti-TAT antibody) candidate to be evaluated at a concentration of 0.04 to 0.4 ⁇ g/mL was mixed and incubated with each inhibitory antigen (prothrombin (manufactured by Enzyme Research Laboratories), thrombin, antithrombin, synthetic TAT complex).
  • inhibitory antigen prothrombin (manufactured by Enzyme Research Laboratories), thrombin, antithrombin, synthetic TAT complex).
  • Those candidate antibodies including a group of inhibited antibodies, were used as primary antibodies and allowed to bind to synthetic TAT complexes immobilized onto 96-well plates.
  • a peroxidase-labeled anti-mouse immunoglobulin antibody manufactured by Dako
  • Dako peroxidase-labeled anti-mouse immunoglobulin antibody
  • the percentage of residual antibody in each antibody was calculated for each concentration of each antigen.
  • Pro-T represents prothrombin
  • T represents thrombin
  • AT represents antithrombin.
  • the amount of the TAT antigen required to achieve an inhibition rate corresponding to the percentage of residual antibody obtained by inhibition with antithrombin at 50 ⁇ g/mL was calculated and compared with the amount of antithrombin to obtain the difference (fold increase) in the reactivity of each antibody to TAT relative to that to antithrombin.
  • a larger difference in the reactivity of an antibody indicates the higher specificity of the antibody to TAT relative to that to antithrombin.
  • the antibodies against the antithrombin part evaluated by indirect inhibition ELISA in Example 4 (TAT-1, TAT-2, TAT-3, TAT-4, and TAT-5) and the antibody against the thrombin part obtained in Example 3 (T-1) were used in sensitization of latex particles and the resulting particles were used for the evaluation of reactivity.
  • Latex particles were sensitized with each antibody by allowing polystyrene latex particles with a size of 0.32 ⁇ m to adsorb the antibody, blocking them with 0.3% BSA solution, subsequently spinning down and washing them with a 0.05% solution of sodium azide (manufactured by Kishida Chemical Co., Ltd.), and then dispersing them again in a 0.05% solution of sodium azide.
  • Reagents containing the latex particles as produced above and sensitized with each antibody were prepared and used for the evaluation of reactivity to TAT.
  • the compositions of the used reagents are as indicated below.
  • For the first reagent a composition of 100 mM Bis-Tris (manufactured by Dojindo Laboratories), pH 6.0, 500 mM NaCl, and 0.15% BSA was used.
  • For the second reagent particles sensitized with each antibody were diluted with 0.05% sodium azide to an absorbance of 1.0 at a wavelength of 700 nm and mixed for use.
  • serum was diluted with saline containing 0.5% BSA and analyzed with the CLEIA reagent to determine the concentration of TAT complexes and the dilution was used as the serum TAT fraction.
  • the analyzed serum TAT fraction was diluted with pooled plasma (manufactured by VitroLogic) to a TAT concentration of 1000 ng/mL and the resulting dilution was used as a measurement sample.
  • the analyzer 7170S (manufactured by Hitachi High-Technologies Co.) was used as a measuring apparatus. Measurement parameters were set to 12 ⁇ L of sample volume, 90 ⁇ L of the first reagent, 90 ⁇ L of the second reagent, 570 nm of main wavelength, and 800 nm of subsidiary wavelength. The absorbance at the 34th photometric measurement point was subtracted by the absorbance at the 20th photometric measurement point and then multiplied by 10000 to determine ⁇ Abs and thereby the measurement was completed.
  • TAT-1, TAT-2 and TAT-3 shown in Table 3 the latex agglutination assay successfully confirmed the high reactivity of those antibodies. Moreover, also in the antibodies TAT-4 and TAT-5, a certain level of reactivity was confirmed, though lower than those of TAT-1, TAT-2 and TAT-3.
  • the cross-reactivity to antithrombin in the prepared reagents was evaluated using samples prepared as described in Example 5 by diluting human serum with saline containing 0.5% BSA to a concentration of 1000 ng/mL and further supplemented with antithrombin to a concentration of 250 or 500 ⁇ g/mL.
  • the reactivity obtained when no antithrombin was added was compared with the reactivity obtained when antithrombin was added at each concentration.
  • any of the measuring reagents, measuring apparatus, and measurement parameters were similar to those in Example 4.
  • the anti-TAT antibodies used for the evaluation are TAT-1, TAT-2, TAT-3, TAT-4 and TAT-5.
  • the evaluation results of the cross-reactivity of the latex reagents to antithrombin are shown in FIG. 5 .
  • the antibodies that achieved a 100-fold or more specificity in the indirect inhibition ELISA of Example 4 retained not less than 70% of the original reactivity even in the case of adding antithrombin to a concentration of 500 ⁇ g/mL.
  • TAT-4, TAT-5 extreme reduction in reactivity due to the increased antithrombin concentration was observed ( FIG. 5 ).
  • composition of the reagents a composition of 100 mM Bis-Tris or MES (manufactured by Dojindo Laboratories), 700 mM NaCl, 0.15% BSA, 0.20% sodium alginate (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.05% EMULGEN-150 (manufactured by Kao Co.) was used for a first reagent, while TAT-1 and T-1 were used as antibodies against the antithrombin part and the thrombin part, respectively, for a second reagent with diluting particles sensitized with each antibody in 0.05% sodium azide to an absorbance of 1.0 at a wavelength of 700 nm and mixing them. Sensitization of latex particles with each antibody was performed similarly to that in Example 5 except that particles with a particle size of 0.20 ⁇ m were used.
  • saline containing 0.5% BSA referred to as Saline
  • Plasma human pooled plasma
  • the human serum analyzed with the CLEIA reagent was diluted with the above-described pooled plasma to each concentration (10, 50, 100 ng/mL) and the resulting dilutions were used as TAT samples.
  • the measuring apparatus and measurement parameters were similar to those in Example 4.
  • a signal ( ⁇ Abs) of not less than 100 at a TAT concentration of 50 ng/mL was used as an indicator and then a pH of not more than 6.6 was found to be preferable for the reaction liquid ( FIG. 7 ).
  • the upper limit of the pH was 6.6 and particularly preferably 6.2 in the viewpoint of the maintenance of reactivity.
  • reagents prepared as described below were used for the composition of the reagents.
  • a reagent containing 100 mM Bis-Tris, pH 6.2, 700 mM NaCl, 0.05% EMULGEN-150, 0.20% sodium alginate, and 0.15% BSA was used as a first reagent.
  • a second reagent similar to that in Example 7 was used.
  • the CLEIA reagent was used as a control reagent for the measurement.
  • the TAT calibrator manufactured by LSI Supremece Co.
  • the TAT calibrator was used as a standard along with the latex reagent or the CLEIA reagent.
  • Example 4 For the samples to be used, 20 samples of citrated plasma were used. To perform the measurement, any of the measuring apparatus and measurement parameters were similar to those in Example 4.
  • the STACIA analyzer manufactured by LSI Supremece Co. was used as a measuring apparatus for the CLEIA reagent and the measurement was performed in accordance with the parameters described in the package insert.
  • the latex reagent according to the present invention indicated a good correlation with the assay reagent according to a conventional CLEIA reagent within the range from 3 ng/mL to 120 ng/mL even in the case of using clinical samples.
  • the use of the present invention enables highly sensitive measurement of clinical samples without treatments such as B/F separation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US15/562,839 2015-03-31 2016-03-29 Reagent and method for assaying thrombin-antithrombin complex Abandoned US20180106793A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
JP2015-074168 2015-03-31
JP2015-074173 2015-03-31
JP2015074168A JP6594641B2 (ja) 2015-03-31 2015-03-31 トロンビン・アンチトロンビン複合体の測定試薬及び測定方法
JP2015074173A JP6615474B2 (ja) 2015-03-31 2015-03-31 トロンビン・アンチトロンビン複合体の測定試薬及び測定方法
PCT/JP2016/060261 WO2016159015A1 (ja) 2015-03-31 2016-03-29 トロンビン・アンチトロンビン複合体の測定試薬及び測定方法

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2016/060261 A-371-Of-International WO2016159015A1 (ja) 2015-03-31 2016-03-29 トロンビン・アンチトロンビン複合体の測定試薬及び測定方法

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/861,040 Division US11237157B2 (en) 2015-03-31 2020-04-28 Reagent and method for assaying thrombin-antithrombin complex

Publications (1)

Publication Number Publication Date
US20180106793A1 true US20180106793A1 (en) 2018-04-19

Family

ID=57005837

Family Applications (2)

Application Number Title Priority Date Filing Date
US15/562,839 Abandoned US20180106793A1 (en) 2015-03-31 2016-03-29 Reagent and method for assaying thrombin-antithrombin complex
US16/861,040 Active US11237157B2 (en) 2015-03-31 2020-04-28 Reagent and method for assaying thrombin-antithrombin complex

Family Applications After (1)

Application Number Title Priority Date Filing Date
US16/861,040 Active US11237157B2 (en) 2015-03-31 2020-04-28 Reagent and method for assaying thrombin-antithrombin complex

Country Status (7)

Country Link
US (2) US20180106793A1 (ja)
EP (1) EP3279659B1 (ja)
KR (1) KR102520342B1 (ja)
CN (2) CN107533050A (ja)
ES (1) ES2908437T3 (ja)
HK (1) HK1244537A1 (ja)
WO (1) WO2016159015A1 (ja)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11378577B2 (en) 2015-10-30 2022-07-05 Lsi Medience Corporation Reagent and method for measuring thrombin-antithrombin complex

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108709988A (zh) * 2018-04-10 2018-10-26 波音特生物科技（南京）有限公司 一种新型抗原抗体反应增敏剂
CN111239403B (zh) * 2019-02-13 2020-12-08 武汉生之源生物科技股份有限公司 一种β2微球蛋白胶乳增强免疫比浊试剂盒及应用
CN111190003A (zh) * 2020-03-30 2020-05-22 吉林省富生医疗器械有限公司 一种视黄醇结合蛋白检测试剂盒及其制备方法
CN116162165A (zh) * 2022-11-22 2023-05-26 杭州博茵生物技术有限公司 抗人凝血酶-抗凝血酶复合物抗体、制备方法、检测试剂及应用

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU628960B2 (en) 1989-04-07 1992-09-24 Teijin Limited Method of immunologically assaying human thrombin- antithrombin III complex, assay reagent and kit therefor
JPH0348158A (ja) * 1989-04-07 1991-03-01 Teijin Ltd ヒト・トロンビン・アンチトロンビン３複合体の免疫学的測定方法、そのための測定試薬およびキット
JPH07238099A (ja) * 1994-02-25 1995-09-12 Dai Ichi Pure Chem Co Ltd モノクローナル抗体及びこれを用いる免疫学的測定法
JP3328053B2 (ja) * 1994-03-16 2002-09-24 積水化学工業株式会社 免疫凝集反応による抗体又は抗原の濃度定量法
JP3618797B2 (ja) * 1994-10-11 2005-02-09 積水化学工業株式会社 免疫測定法
JPH1026621A (ja) * 1996-07-12 1998-01-27 Dai Ichi Pure Chem Co Ltd イムノアッセイ法
JP2001228153A (ja) 2000-02-15 2001-08-24 Dai Ichi Pure Chem Co Ltd 免疫学的測定方法
JP4390963B2 (ja) * 2000-04-05 2009-12-24 シスメックス株式会社 免疫測定方法及び試薬キット
JP2002316999A (ja) 2001-04-17 2002-10-31 Dai Ichi Pure Chem Co Ltd モノクローナル抗体

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11378577B2 (en) 2015-10-30 2022-07-05 Lsi Medience Corporation Reagent and method for measuring thrombin-antithrombin complex

Also Published As

Publication number Publication date
CN115327121A (zh) 2022-11-11
EP3279659A1 (en) 2018-02-07
US20200256864A1 (en) 2020-08-13
EP3279659A4 (en) 2018-11-14
EP3279659B1 (en) 2022-02-16
US11237157B2 (en) 2022-02-01
WO2016159015A1 (ja) 2016-10-06
KR102520342B1 (ko) 2023-04-10
CN107533050A (zh) 2018-01-02
HK1244537A1 (zh) 2018-08-10
KR20170132310A (ko) 2017-12-01
ES2908437T3 (es) 2022-04-29

Similar Documents

Publication Publication Date Title
US11237157B2 (en) Reagent and method for assaying thrombin-antithrombin complex
EP3370064B1 (en) Reagent and method for measuring thrombin-antithrombin complex
KR101669918B1 (ko) 사람 체액 중의 시스타틴 ｃ의 측정방법
JP6110786B2 (ja) Ｐｓａの測定方法及びその試薬
JP5860394B2 (ja) 新規モノクローナル抗体ならびにｄダイマーの免疫学的測定法
JP6594641B2 (ja) トロンビン・アンチトロンビン複合体の測定試薬及び測定方法
JP6615474B2 (ja) トロンビン・アンチトロンビン複合体の測定試薬及び測定方法
JP6687183B2 (ja) トロンビン・アンチトロンビン複合体の測定試薬及び測定方法
JP6037043B2 (ja) ＴＲＡＣＰ−５ｂ（酒石酸抵抗性酸性フォスファターゼ５ｂ）に特異的なタンパク定量法
JP7054378B2 (ja) Ｃａ１９－９測定方法及びｃａ１９－９測定キット、並びに、これらに用いる抗体固定化担体及びその製造方法
JP7355140B2 (ja) セリンプロテアーゼの検出用または測定用試薬
KR20240151234A (ko) 세린 프로테아제의 검출용 또는 측정용 시약
WO2013084504A1 (ja) 高血圧症の予知方法
JP5585587B2 (ja) ５．９ｋＤａペプチドの免疫学的測定方法
JPWO2019039557A1 (ja) ジカウイルスを検出する方法及びキット

Legal Events

Date Code Title Description
AS Assignment

Owner name: LSI MEDIENCE CORPORATION, JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:YOSHIDA, TATSUYA;YANG, YUHANG;REEL/FRAME:043731/0276

Effective date: 20170911

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION