Simultaneous detection of cluster of differentiation markers on leukemia-derived exosomes by multiplex immuno-polymerase chain reaction via capillary electrophoresis analysis.

Acute myeloid leukemia (AML) is a heterogeneous disease, and there are critical interests in detecting multiple biomarkers as a single biomarker detection cannot reflect the exact phase of the disease. Exosomes derived from different types of AML cells contain respective combinations of cluster of differentiation (CD) that may be used to guide the molecular typing of AML in clinic. Here, aiming to build more precise molecular typing of AML, we demonstrate multiplex immuno-PCR (mI-PCR) assay for simultaneous detection of multiple surface CDs of exosomes of AML via capillary electrophoresis with laser induced fluorescence (CE-LIF). This method comprises of four steps: 1) chemical attachment of reporter DNA sequence to the specific detection antibodies; 2) binding of the detection antibodies to their targets of the exosomes; 3) DNA amplification of the reporter DNA; and 4) capillary electrophoresis analysis of the PCR products. With the method, we first realized simultaneous detection of 5 target CD molecules (CD9, CD34, CD63, CD123, c-Kit/CD117, FLT-3/CD135) on leukemia cell-derived exosomes with high detection sensitivity. The limit of detection (LOD) and limit of quantification (LOQ) are 2.41±0.04 particles/µL and 8.02±0.16 particles/µL respectively for leukemia cell-derived exosomes. This mI-PCR is found sensitive enough to detect picogram (10-12) levels of protein concentrations with high recovery (95%) in spiked serum sample experiments. We thus anticipate that the proposed method is promising in sensitive detection of multi-target to assist precise molecular typing of many complex diseases.