Establishment Of An Enzyme-linked Immunosorbent Assay For Measurement Of Von Willebrand Factor Ristocetin Cofactor Activity And Its Applicatin In Clinic

Background:Von Willebrand factor (vWF) is a large multimeric adhesive glycoprotein with complex roles in thrombosis and hemostasis. The first is to bridge platelets to the blood vessel wall forming a link between glycoprotein (GP)Ⅰb on the surface of the platelet with collagen and the underlying subendothelium, and the second is to act as a carrier protein for factorⅧ(FⅧ) to protects FⅧfrom rapid proteolytic degradation. The deficiency or abnormal function of vWF causes von Willebrand's disease (vWD).von Willebrand's disease (vWD) is the most common heterogeneous bleeding disorder affecting 0.1～1% of the general population. The prevalence of symptomatic cases requiring occasional transfusion treatment is about one in 10,000 persons. Patients with von Willebrand's disease may have normal levels of von Willebrand factor (vWF) antigen, it is therefore important to measure not only the antigen concentration but also the vWF activity. The most widely used method for measurement of vWF activity is the ristocetin cofactor assay (vWF: RCof), which is crucial for the laboratory diagnosis, classification and management of von Willebrand's disease (vWD). In our country, the vWF: RCof acitivity is determined by using a platelet agglutination procedure employing a platelet aggregometer, but the vWF: RCof agglutination test has an unacceptable high inter-assay and intra-assay variability. In 2000, Vanhoorelbeke et al described a new type of ELISA-based method in which no longer utilizes platelets but is based on the use of a recombinant fragment of GPIbα(rfGPIbα, His1-Val289) and an anti-GPIba monoclonal antibody (2D4), this ELISA assay for vWF: RCo showed a low inter-assay and intra-assay variability than that of the agglutination method. In this study a simple and reliable enzyme-linked immunosorbent assay was established for the determination of von Willebrand factor activity, utilizing an anti-GPIba monoclonal antibody (SZ-151) developed in our own laboratory, the test would be commercially available in the near future. This ELISA assay that could easily and accurately help in diagnosing and classifying vWD especially the type 2 vWD among Chinese would be of great use.Objective:(1) To prepare a monoclonal antibody (mAb) against human platelet glycoprotein Ibαwhich could be useful for assays of plasma von Willebrand factor ristocetin cofactor activity(vWF: Rcof) and can be used for diagnosing patients with vWD.(2) To prepare and purify the recombinant fragment of GPIbα(His1-Val289) (rfGPIbα).(3) To develop a vWF: RCof ELISA test with the anti-GPIba mAb SZ-151 and the rfGPIbαHis1-Val289 and measure the sensitivity and repeatability of the method.(4) To apply the vWF: RCof ELISA in measuring plasma sample of the patient with vWD and evaluate its value in clinic.Methods:(1) A monoclonal antibody (mAb) against human platelet GPIbαwhich does not inhibit vWF binding to GPIba was developed using a classical method and named SZ-151.(2) The CHO Cells expressing rfGPIbαHis1-Val289 which having an Fc region was cultured and the rfGPIbαHis1-Val289 was purified with affinity chromatography column. Its purity, immune activity and biological activity were identified with the biochemical methods.(3) Establishment of vWF: Rcof-ELISA: A monoclonal antibody (mAb) to GPIbα, SZ-151 was immobilized onto microtiter plate wells. Plates were blocked overnight followed by incubation with a solution containing rfGPIb, and then platelet-poor plasma (PPP) and ristocetin was added to the wells, respectively. The rfGPIbαbound to SZ-151, in turn, captured plasma vWF in the presence of ristocetin; finally, bound vWF was detected by a polyclonal anti-vWF antibody conjugated with horseradish peroxidase (HRP). Visualisation was obtained with tetramethylbenzidine (TMB) and the absorbance was determined at 450 nm.(4) The plasma vWF: RCof in 36 normal donors and 25 vWD patients was tested using the vWF: Rcof-ELISA method. The sensitivity and repeatability of the test were detected. The new ELISA method was evaluated in our laboratory in a blind comparison with the previously available vWF: RCof ELISA coated-plate with mAb 2D4 and explored the correlation of this assay with vWF: Ag and vWF: CB.Results:(1) A murine mAb against human platelet membrane glycoprotein GPIb was developed and denominated as SZ-151. ELISA showed that SZ–151 did not inhibit plasma von Willebrand factor ristocetin cofactor activity.(2) 2 mg recombinant fragment in culture supernatants per liter could be purified with affinity chromatography.(3) Level of sensitivity:The detection limit (DL) of our vWF: RCof-ELISA was 0.01 U/ dL (1/12800 plasma dilution).(4) Repeatability: The inter- and intra-assay variability (CV) was 8% and 12% respectively.(5) Clinical applications of the vWF: Rcof- ELISA The mean level of vWF:RCof was 91.5 U/dL(47.3-169.2 U/dL) in controls (n = 36), 31.8 U/ dL(22.3-56.9 U/dL) in type 1 vWD patients (n = 5), 4.8U/ dL(0.6-11.8U/dL) in type 2A (n = 9), 8.6U/dL(1.6-19.7U/dL) in type 2B (n = 4), 3.9 U/ dL (1.0-6.8U/dL) in type 2M (n = 2)and 1.0U/dL(0.5-1.6U/dL) in type 3 (n = 5).(6) Comparison of Methods The correlation of vWF: Rcof-ELISA results between the SZ-151 caoted-plate and 2D4 coated-plate was concordant (r=0.96). Moreover, the values were correlated well with those obtained from the vWF: Ag (r = 0.91) and vWF: CB(r = 0.85).Conclusions:(1) A mAb, SZ-151 against platelet glycoprotein Ib was developed, which could be useful in assays of plasma von Willebrand factor ristocetin cofactor activity(vWF:Rcof).(2) The purified rfGPIbαhas higher purity and biology activity.(3) An ELISA method to measure ristocetin cofactor activity was established. This assay is more sensitive and precise than the previously published assays.(4) The plasma vWF: RCof in 36 normal donors and 25 patients with different types of vWD was measured, and these results corresponded with the vWF: CB and the vWF: Ag values.(5) This test can routinely be used in laboratories to diagnose and identify vWD subtypes in combination with other assays and to offer great advantages for correct type 2B vWD diagnosis.