Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material

Abstract

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 μl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.

Fig. 1.

Schematic description of multiplex homogenous proximity ligation assay. (1) The samples are incubated with 23 pairs of proximity probes in 1 μl sample. (2) Upon proximal binding of the pairs of PLA probes to a common antigen the oligonucleotide sequences are ligated using a connector oligonucleotide. (3) The ligated products are amplified in multiplex using all pre-amplification primers simultaneously. (4) This is followed by quantification of each biomarker by quantitative real time PCR using only the unique primer pair for a given analyte.

Fig. 2.

Evaluation of assay performance for 4 × 24-plex homogenous PLA. Exemplified by data for the detection of interleukin 6(IL-6) where the recombinant antigens are present at concentrations ranging from 2–200 pm in buffer. To illustrate specificity, assay evaluation was also performed in smaller antigen subset mixes selected from all antigens in a panel for which IL-6 was only present in mix #3. Assay specificity was also assessed in 100% chicken plasma. Linearity of the assay was further tested by performing two fivefold dilutions in PBS with 0.1% BSA from 100% to 4% plasma and recovery experiments calculated by spiking 20 pm pure antigen in plasma, data normalized against GFP.

Fig. 3.

Estimated assay sensitivity by back-calculating from the signal over background at the point of the standard curve nearest background (0 pm) value for the 50 analytes included in panel 1–4 for which antigen was available. Values shown ranged from 26 fM up to 110 pm.

Fig. 4.

Variable importance for the 54 assays used in random forest classification in 74 CRC patients and 74 matched healthy controls. Importances in 15 individual random hold-outs are shown as dots and the median value as a circle.