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) markers that may be used to guide the molecular typing of AML in the 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 on 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 on 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 five target CD molecules (CD9, CD34, c-Kit/CD117, CD123, and 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 multitargets to assist in the precise molecular typing of many complex diseases.