Establishment of Double Antibody Sandwich ELISA for the Determination of Human Soluble VE-Cadherin in Human Plasma and Its Application

Abstract

To establish double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of human soluble VE-cadherin in human plasma and to investigate its value in clinical use. The monoclonal antibodies against human VE-cadherin were prepared from BALB/c mice immunized with prokaryotic expression recombinant proteins, and the best combination of double antibodies was selected by checkerboard titration method. Double antibody sandwich ELISA for the determination of human VE-cadherin was established by using HRP-labeled McAb as a detection antibody and a capture antibody. The methodology performance was evaluated. The plasma concentrations of VE-cadherin in 28 healthy subjects and 60 patients with cancer were determined. The double antibody sandwich ELISA for the determination of human VE-cadherin was established by selecting the combination of double antibodies. The detection limit was 24.7 pg/ml, the coefficients of variation for inner-batch and inter-batch were 4.1%-7.7% and 8.7%-10.8% respectively. The average recovery was 96.7%. The plasma level of soluble VE-cadherin in normal controls was 262.1±11.75 pg/ml. The plasma level of soluble VE-cadherin was 173.9±17.98 pg/ml in 24 patients with leukemia, 311.7±25.24 pg/ml in 14 patients with stomach cancer, 206.8±25.01 pg/ml in 11 patients with lung cancer, and 310.7±11.82 pg/ml in others patients(9 patients with breast cancer, 1 patients with gliomas, 1 patients with liver cancer). The developed ELISA kit has better sensitivity and specificity, and can be used in detection of human soluble VE-cadherin in human plasma, therefore, it can provide a new mathod for diagnosis of cancer patients.