Methods and kits for measuring ADAMTS13/FXI complexes

a technology of adamts13 and complexes, which is applied in the field of adamts13 activity measurement and diagnosis of thrombocytopenia purpura, can solve the problems of affecting the clinical presentation of patients with ttp, the clinical manifestation of ttp is difficult to distinguish from hemolytic uremic syndrome, and the clinical manifestation of ttp is difficult to distinguish from ttp hemolytic syndrome, so as to reduce the amount of adam

Inactive Publication Date: 2006-04-06
AMERICAN DIAGNOSTICA
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AI Technical Summary

Problems solved by technology

Decreased VWF cleaving protease activity leads to persistence of unusually large multimers of VWF that bind to platelets, causing platelet aggregates, microangiopathic hemolysis, and thrombocytopenia in patients with TTP.
Clinical manifestations of TTP are difficult to distinguish from hemolytic uremic syndrome (HUS), another thrombotic microangiopathic disorder.
In addition, obtaining results can take several days using available tests, which is detrimental to patients presenting with TTP, who require rapid diagnosis.
Attempts at developing such an assay using ADAMTS13 isolated from plasma have not been successful.
These results demonstrate the difficulty in the art of developing a rapid, reliable assay for ADAMTS13 activity.

Method used

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  • Methods and kits for measuring ADAMTS13/FXI complexes
  • Methods and kits for measuring ADAMTS13/FXI complexes
  • Methods and kits for measuring ADAMTS13/FXI complexes

Examples

Experimental program
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Effect test

example 1

Measurement of ADAMTS13 Activity in PRP Using LVY-pNA and LVY-AMC Peptidyl Substrates

[0114] As discussed above, Furlan et al. reported that some peptidyl-pNA moieties, such as XLY-pNA and XVY-pNA, were not substrates for ADAMTS13. We prepared LVY-pNA and tested it for its ability to be cleaved by ADAMTS13. The results shown in FIG. 2 confirm the findings of Furlan et al., i.e., LVY-pNA was not cleaved by ADAMTS13.

[0115] Thus, the nature of the leaving group of the peptidyl substrate appears to be important in determining the ability of ADAMTS13 to cleave a chromophor or a fluorophor from the end of a peptidyl substrate.

example 2

Measurement of ADAMTS13 Activity in PRP Using Peptidyl Substrates of Different Lengths

[0116] In order to determine the effect of modifying the length of the peptide sequence conjugated to the AMC fluorochrome, normal PRP (20 μl) was incubated at 37° C. with a final concentration of 0.8 mM Suc-LLVY-AMC or LVY-AMC in 10 mM Tris-HCl pH 8.0 buffer in total volume of 200 μl. Fluorescence was measured using a microtiter fluorophotometric plate reader (Ex 360 nm / Em 460 nm). Both peptidyl-AMC substrates were cleaved by ADAMTS13 in PRP, with the Suc-LLVY-AMC giving a higher signal over time as compared to the LVY-AMC substrate (FIG. 3).

[0117] This example demonstrates that different ADAMTS13 substrates can be made by altering the peptide sequence that is conjugated to the AMC fluorophor.

example 3

Measurement of ADAMTS13 Activity Using a FRET Substrate

[0118] ADAMTS13 activity was measured using a fluorescent substrate attached to a donor moiety and an acceptor moiety that functions by fluorescence resonance energy transfer (FRET). The ADAMTS13 selective substrate, NH2-Arg-Lys(DABCYL)-NLVYMVTGD(EDANS)-Arg-COOH, was used in this example. The NLVYMVTGD peptide sequence of this substrate is homologous to the ADAMTS13 cleavage site on VWF. The intact peptidyl substrate has low fluorescence, due to quenching of the DABCYL-fluorochrome by the EDANS-quencher. Cleavage of the substrate between the tyrosine and methionine of the peptide sequence results in an increase in fluorescence.

[0119] Isolated platelets in 10 mM Tris-HCl pH 8.0 assay buffer were incubated with a final concentration of 8 μg / ml of the FRET substrate at 37° C. The fluorescence was measured in a spectrofluorometric plate reader (Ex 360 nm / Em 440 nm). FIG. 4 shows that incubation of platelets with NH2-Arg-Lys(DABCYL...

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Abstract

Provided are assays and kits to detect ADAMTS13 activity using peptide substrates and ADAMTS13 / Factor XI complexes using ELISA. These assays and kits can be used for diagnostic applications and to evaluate treatment of thrombotic thrombocytopenic purpura (TTP). Also provided is a novel form of ADAMTS13 found on platelets.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. patent application Ser. No. 10 / 894,702, filed Jul. 19, 2004. This application makes reference to International Application No. PCT / US2004 / 023177, also filed Jul. 19, 2004. [0002] This application and each of the documents cited in this application (“application cited documents”), and each document referenced or cited in the application cited documents, either in the text or during the prosecution of this application, as well as all arguments in support of patentability advanced during such prosecution, are hereby incorporated herein by reference. Various documents are also cited in this text (“application cited documents”). Each of the application cited documents, and each document cited or referenced in the application cited documents, is hereby incorporated herein by reference.FIELD OF THE INVENTION [0003] The invention provides assays for measurement of ADAMTS13 activity and diagnosi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/537G01N33/543C07K14/745
CPCC12Q1/37C12Q2337/00
Inventor GREENFIELD, ROBERTGUINTO, ENRIQUETARANZURMAL, SAFIGRAFOS, NICHOLAS
Owner AMERICAN DIAGNOSTICA
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