Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Lateral-Flow Immunochromatographic Strip for the Detection of Digoxin in Human Blood

Abstract

Mouse-mouse hybridoma cell lines producing stable, highly specific monoclonal antibodies with good affinity for the cardiac glycoside digoxin (DIG) were established to construct an indirect enzyme-linked immunosorbent assay and lateral-flow immunochromatographic strip to detect DIG in human blood. The hapten DIG was coupled to bovine serum albumin or chicken ovalbumin by sodium periodate oxidation. The highest sensitivity and specificity antibody had a median inhibitory concentration (IC₅₀) of 0.45 ng/mL, a linear range of detection of 0.293-0.7 ng/mL, and low cross-reactivity with several DIG analogues. The cut-off value of the lateral-flow immunochromatographic strip was 5 ng/mL when the strip was tested with human blood. The immunochromatographic lateral flow strip test provides a quick and convenient method for determining DIG in plasma which can be visually observed in only 5 min to promote rational drug use.

Figure 1

Synthesis of the DIG hapten.

Figure 2

Electrophoresis of different antigens. (a) 1: BSA, 2: DIG–BSA and (b) 1: OVA, 2: DIG–OVA.

Figure 3

Standard curve obtained by ic-ELISA based on antibody 1H3.

Figure 4

Detection of three anti-DIG cell lines 1H2, 1H3, and 2D12 by lateral-flow immunochromatographic assay (ICA) strip in PBS sample (1 = 0 ppb, 2 = 0.5 ppb).

Figure 5

DIG detection by lateral-flow ICA strip in PBS sample (a) and human blood sample (a) 1 = 0 ng/mL, 2 = 0.25 ng/mL, 3 = 0.5 ng/mL, 4 = 1 ng/mL, 5 = 2.5 ng/mL, and 6 = 5 ng/mL; the cut-off value for DIG was 5 ng/mL. (b) 1 = 0 ng/mL, 2 = 0.1 ng/mL, 3 = 0.25 ng/mL, 4 = 0.5 ng/mL, 5 = 1 ng/mL, 6 = 2.5 ng/mL and 7 = 5 ng/mL; the cut-off value for DIG was 5 ng/mL.