A Rapid, Simple, and Low-Cost CD4 Cell Count Sensor Based on Blocking Immunochromatographic Strip System.

The counting of CD4+ T lymphocytes (CD4 cells) is a critical test for evaluating the immune function of HIV-infected peoples and tumor patients. A rapid, simple, accurate, and low-cost CD4 cell counting method as a diagnostic tool is increasingly required in the clinic. We designed and developed a novel fluorescent immunochromatographic strips (ICS) system based on the blocking principle for counting CD4 cells. The strategy of this system is to count CD4 cells indirectly, by measuring the free CD4 antibodies that were not bound by CD4 cells. The fluorescent antibodies bound to CD4 cells were blocked at the filter pads, resulting in a decrease in fluorescence of free CD4 antibodies measured. The number of CD4 cells was inversely related to the fluorescence intensity. The CD4 count-ICSs exhibited a quasilinear response ( R2 = 0.96) to logarithmic CD4 cell concentrations in PBMC samples in the range of 50-1000 cells/µL. In addition, the CD4 count-ICSs reliably quantified CD4 cells in whole blood samples, where the assay exhibited a linear correlation ( R2 = 0.976) readout for CD4 cell concentrations ranging from 100 to 800 cells/µL. To validate the clinical applicability of this method, 54 blood samples were measured: the detection results showed a high correlation ( R2 > 0.97) with the flow cytometry (FCM) analysis. The fluorescent ICSs can be used to count CD4 cells in blood samples, which have a high coincidence rate with FCM analysis; therefore, the CD4 count ICS system is an excellent candidate method for CD4 cell counting in resource-limited settings.

Inhibition activity of a disulfide-stabilized diabody against basic fibroblast growth factor in lung cancer.

The over-expression of basic fibroblast growth factor (bFGF) plays a crucial role in the development, invasion and metastasis of lung cancer. Therefore, neutralizing antibodies against bFGF may inhibit the growth of lung cancer. In this study, a Disulfide-stabilized diabody (ds-Diabody) against bFGF was constructed by site-directed mutation and overlap extension PCR (SOE-PCR) at the position of VH44 and VL100 in the scFv. The ds-Diabody was constructed and expressed in Pichia pastoris. We found that the ds-Diabody against bFGF could efficiently suppress the proliferation, migration and invasion of human lung cancer A549 cells in vitro. Moreover, in A549 cells, the ds-Diabody against bFGF could inhibit bFGF-induced activation of downstream signaling regulators, such as phospho-Akt and phospho-MAPK. In the nude mouse xenograft model of lung cancer, the ds-Diabody against bFGF could significantly inhibit tumor growth and decrease the densities of micro-vessels and lymphatic vessels in tumor tissue. Our data indicate that the ds-Diabody against bFGF could effectively suppress the lung cancer growth through blockade of bFGF signaling pathway and inhibition of tumor angiogenesis, which may make it a potential therapeutic candidate antibody drug for human lung cancer therapy.