Using SPARCL technology to develop immunoassays for biomarker detection and pharmacokinetic studies

Introduction

Biomarkers are used in diverse scientific fields to evaluate normal and pathogenic processes or the response to therapy. Here we look at the biomarker interleukin 8 (IL-8) which is often associated with inflammation and has been shown to be increased by oxidative stress. Pharmacokinetic (PK) assays are used to measure the concentration of a drug that has been administered to a human or animal. PK assay data are used to evaluate elimination profiles of the drug and are important in drug safety and toxicology studies.

In this application note, we will show how the performance of the FLUOstar Omega microplate reader enables the detection of SPARCL (Spatial Proximity Analyte Reagent Capture Luminescence) assays. Examples of biomarker detection and pharmacokinetic assays are presented.

Assay Principle

SPARCL detection technology was used for rapid immunoassay development. This technology is a proximity-dependent and homogeneous method where an antibody/antigen interaction leads to the proximity of a chemiluminescent substrate (acridan, bound to antibody 1)) and an oxidative enzyme (horseradish peroxidase (HRP, bound to antibody 2)). The introduction of a trigger solution containing H₂O₂ generates a flash of light proportional to the amount of analyte present (Figure 1).

Materials & Methods

• SPARCL kits (Lumigen)
• FLUOstar Omega microplate reader (BMG LABTECH)
• 96-well, white, flat bottom microplates from Greiner
• Other reagents were obtained from commercial sources

A typical Lumigen SPARCL assay workflow is shown below.

For each experiment, one antibody was labeled with acridan according to SPARCL kit instructions while a second HRP-labeled antibody was purchased.

Biomarker Assay
A 1:2 dilution series of IL-8 was prepared from a starting concentration of 1000 pg/mL. Dilution of controls and the dilution series were made in cell culture media or PBS containing 0.1% BSA and 10% human plasma.

Pharmacokinetic Assay
A standard curve was generated using a dilution series of human IgG in neat (undiluted) rat serum. The highest point in the standard curve was 1000 ng/mL. Quality Control samples (QC) were made by spiking human IgG drugs into a neat rat matrix. QC concentration values were interpolated from the standard curve. The interpolated QC sample values were used to determine accuracy, precision, and total error.

For both experiments sample plating, antibody addition, incubation, and addition of background reducing agent were performed according to instructions.¹ The plates were then read on the FLUOstar Omega plate reader with the following settings which include injection of the trigger solution.

FLUOstar Omega instrument settings

Results & Discussion

The FLUOstar Omega features reading at the time of injection which allows for the assessment of signal produced in the SPARCL reaction upon addition of the trigger solution (Figure 2).