Substrate, method and kit for in-vitro quantitative determination of activity of vWF-CP enzyme

Abstract

The invention relates to the biological field, and especially relates to a substrate, a method and a kit for in-vitro quantitative determination of the activity of vWF-CP enzyme. The substrate comprises a vWF functional area amino acid sequence. The end N and the end C of the vWF functional area amino acid sequence are respectively combined with a HIS label amino acid sequence and a FLAG label amino acid sequence. The method comprises the following steps: the holes of an ELISA plate are coated with an antibody in advance; and then, the substrate, a sample to be tested, an ELISA antibody, and chromogenic agent are added, and the absorbance of liquid in the holes of the ELISA plate is measured using an ELISA reader. The kit comprises an ELISA plate, a substrate, diluent, washing liquid, buffer solution, an enzyme-labeled antibody, chromogenic solution and stop solution, or comprises an ELISA plate, an antibody, a substrate, diluent, washing liquid, buffer solution, an enzyme-labeled antibody, chromogenic solution and stop solution. The substrate, the method and the kit for in-vitro quantitative determination of the activity of vWF-CP enzyme have the advantages of high sensitivity, high accuracy, simple operation and low price, and are suitable for broad patient screening.

Description

technical field [0001] The invention relates to the field of biology, in particular to a substrate, method and kit for quantitatively measuring vWF-CP enzyme activity in vitro. Background technique [0002] Thrombotic Thrombocytopenic Purpura (TTP) is a severe disseminated thrombotic microangiopathy characterized by microangiopathic hemolytic anemia, decreased consumption of platelet aggregation, and microthrombosis causing organ damage (such as the kidney, central nervous system, etc.) According to early reports, the incidence of TTP is 0.2 to 1 / 100,000. With the gradual recognition of this disease in clinical practice, the incidence of TTP has an upward trend in recent years. At present, no relevant epidemiological statistics have been seen in China. Most of the patients are women, and the disease can occur at any age, but the most common age of onset is mostly 20 to 60 years old, and there is no regional or racial difference. In clinical practice, patients with typical...

AI Technical Summary

Problems solved by technology

Most of the previously reported literature used GST-vWF73-His as a substrate, (Zhou W, Tsai HM. An enzyme immunoassay of ADAMTS13distinguishes patients with thrombotic thrombocytopenic purpura from normal individuals and carriers of ADAMTS13 mutations. Thromb) or a fragment of the A2 region as a substrate ( Haemost 2004;91:806–11Whitelock JL, Nolasco L, Bernardo A, Moake J, Dong JF, Cruz MA. ADAMTS-13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant vWF-A2 domain as substrate. JThromb Haemost 2004;2 :485–91.), the molecular weight of this recombinant protein is relatively large, the testing process is complex and time-consuming, it is difficult to meet the needs of large-scale TTP screening, and it is not suitable for the clinical needs of emergency diagnosis and treatment of patients in the emergency room

Images