Toward next generation plasma profiling via heat-induced epitope retrieval and array-based assays

Abstract

There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care. Here, we used a combination of undirected target selection, antibody suspension bead arrays, and heat-induced epitope retrieval to allow for protein profiling of human plasma in a novel and systematic manner. Several antibodies were found to reveal altered protein profiles upon epitope retrieval at elevated temperatures with limits of detection improving into lower ng/ml ranges. In a study based on prostate cancer patients, several proteins with differential profiles were discovered and subsequently validated in an independent cohort. For one of the potential biomarkers, the human carnosine dipeptidase 1 protein (CNDP1), the differences were determined to be related to the glycosylation status of the targeted protein. The study shows a path of pursuit for large scale screening of biobank repositories in a flexible and proteome-wide fashion by utilizing heat-induced epitope retrieval and using an antibody suspension bead array format.

Fig. 1.

Schematic view of bead array assay. Samples (plasma, serum, urine, etc.) are distributed into microtiter plates (1) following a defined, randomized plate layout. Activated biotin (B; 2) is added to modify the protein content of the samples (3) followed by quenching of remaining labeling reactivity. Antibodies are coupled onto beads with distinct color codes (4), and bead arrays in suspension are created. Labeled samples are heat-treated (5) after being diluted in assay buffer, and without removal of biotin excess, beads and samples are combined and incubated (6). Proteins that have not been captured by the antibodies are washed away (7), and fluorescently labeled streptavidin is added (8) to bind to the biotinylated target protein. The color-coded beads are identified via a green laser, and the concurrently emitted reporter fluorescence, excited by a red laser, allows for the assignment of intensity values (9).

Fig. 2.

Effect of heat treatment on protein profiles. Ninety-six antibodies were used to analyze samples treated at temperature levels between 23 and 96 °C in triplicates. A, the number of antibodies that showed increased (solid line) and decreased signal (dotted line) are shown as compared with the values at 23 °C. B, the results for each antibody are shown, and the intensity -fold changes (log₂) was plotted relative to alterations in comparison with 23 °C.

Fig. 3.

Temperature-dependent antibody capture performance. A, plasma samples spiked with PSA were used to determine the limit of detection with two anti-PSA antibodies, 8A6 (left panel) and 1344 (right panel), treating the samples at 23 and 72 °C. A five-parametric logistic fit was applied to calculate LOD values. B, for samples treated at 23 and 72 °C, the relation between PSA concentrations determined at the clinic and intensity values obtained with three anti-PSA antibodies is shown. Besides 8A6 (gray circles) and 1344 (triangles) being functional with spiked PSA, the antibody 1H12 (black diamonds) allowed resolving difference in PSA levels below 10 ng/ml (see supplemental Table 3). MFI, median fluorescence intensity; AU, arbitrary units.

Fig. 4.

Biomarker discovery at different epitope retrieval temperatures. A, the discovery cohort (supplemental Table 2A) was analyzed at the three treatment temperatures, and the patterns obtained in the heat maps visualize the apparent influence of heat treatment on protein profiles. B, from the comparative study of patient groups, antibodies were identified with significantly different protein profiles (p < 0.01) in a heat treatment-dependent manner as summarized in supplemental Table 4. To highlight some of the results, the findings from a comparison of patient groups with very high (group A) and normal (group B) PSA levels are illustrated in volcano plots. From each temperature, the two most significant findings were selected to show the intensity distribution within the sample groups A and B alongside of sample groups with elevated tPSA (groups C and D). Of the latter groups, samples of group C had a low free-to-total PSA ratio compared with group D. The antibodies represented here target the gene products of CNDP1, FLNB, HCRTR1, TMEM59L, and ATRN. MFI, median fluorescence intensity; AU, arbitrary units. For each patient group the box-and-whisker plots represent intensities within lower and upper quantile (box), the median (horizontal line in box), percentiles of 5% and 95% (whiskers), and outliers (open circles).

Fig. 5.

Validation of potential biomarkers. A, plasma samples from patients in the groups with high (group A) and normal (group B) PSA values were analyzed using Western blot analysis. For anti-HCRTR1 (HPA014018; top), differences in protein abundance appeared for a ±50-kDa variant of HCRTR1. With anti-CNDP1 (HPA008933; middle), evenly strong bands were observed at a molecular mass of ±190 kDa, matching a glycosylated form of CNDP1. Weaker bands found at ±55 kDa most likely represent the non-glycosylated isoform. Plasma analyzed with anti-TMEM59L (HPA010661; bottom), a glycoprotein with an unmodified isoform of 37 kDa, revealed bands at ±55 kDa, and among the four stronger bands, three were from samples of group B. B, a second and independent set of samples composed of patients diagnosed with different phenotypes of prostate cancer and controls was studied. Profiles were age-normalized and appeared to be lower for anti-CNDP1 when aggressive and less aggressive cases were compared. This was contrast to profiles from an antibody targeting PSA (1344). MFI, median fluorescence intensity; AU, arbitrary units. For each patient group the box-and-whisker plots represent intensities within lower and upper quantile (box), the median (horizontal line in box), percentiles of 5% and 95% (whiskers), and outliers (open circles).