Sequential Multiplex Analyte Capturing for the quantification of GCF biomarkers

Sequential Multiplex Analyte Capturing is a technique to repeatedly probe samples with different sets of antibody-coated magnetic beads.

Objectives: To test if Sequential Multiplex could be used to reliably measure analytes in GCF.

Methods: Quality control samples (QC) were prepared using standards provided by 7 Millipore kits which collectively could measure up to 101 analytes.  QCs were used to assess the loss of signal resulting from sequential processing of samples.  Sixty six individual GCF samples from 20 periodontitis and 13 periodontally healthy subjects were also tested.  After overnight incubation with the first set of beads, magnetic beads were immobilized using a magnetic plate.  QC and GCF samples were recovered without disturbing the magnetic beads and incubated with the second set of beads.  The first set of beads was then processed according to protocol to measure the corresponding analytes.  This procedure was repeated until samples were assayed using the 7 kits.  The frequency of detection of all analytes was computed.  Significance of differences between healthy and periodontitis subjects was determined using Mann-Whitney test.  Four analytes were present in both kit 1 and kit 5 and correlation between values from the different kits was tested using Pearson correlation coefficient.

Results: The median frequency of detection for the 101 analytes was 98%, lower quartile 94% and upper quartile 100%.  Transfer of samples across kits did not significantly affect signal intensity.  Values for analytes measured both in kits 1 and 5 presented a high degree of correlation (p<0.001).  Median levels of 29 analytes were significantly different between healthy and periodontitis subjects (p<0.001).

Conclusions: Sequential Multiplex could be used to measure 101 biomarkers in GCF samples.  Some of the analytes detected had never been quantified in GCF.  Of the 29 biomarkers differentially expressed in health and periodontitis, 11 required sequential assays to be detected.