Verifying SPARCL performance on the CLARIOstar equipped for reading at time of injection

Introduction

Since its introduction in the 1970s, the ELISA (enzyme-linked immunosorbent assay) remains a very popular technique to assess the presence or quantity of an analyte.^1,2 Despite the high utility of this approach and the continued advances to improve sensitivity, most ELISA-type assays require washing steps. These steps are time-consuming and tedious or require equipment dedicated to the task.

Immunoassays that use SPARCL from Lumigen provide no-wash detection. We have previously shown the performance of the Omega microplate reader in the detection of this luminescent assay.³ Here we show the CLARIOstar is also excellent for detection of SPARCL immunoassays. Furthermore, we show that the SPARCL assay can be performed in a 384-well format for improved throughput.

Assay Principle

As the SPARCL acronym implies (spatial proximity analyte reagent capture luminescence) it detects an analyte in solution and creates a light signal (Figure 1). It is an immunoassay that employs 2 antibodies: one bound to acridan the other bound to HRP (horseradish peroxidase). The binding of these antibodies to an analyte brings the substrate and enzyme into proximity such that when a trigger solution (H₂O₂) is added a light signal is produced. The intensity of the flash of light is proportional to the amount of analyte present making it a tool for quantifying the analyte.

Materials & Methods

• SPARCL Generic Human IgG Pharmacokinetic Assay Kit (Lumigen)
• CLARIOstar microplate reader (BMG LABTECH)
• White, 96-well plates (Greiner)
• White, 384-well plates (Corning)

Standard curves were prepared using 1:2 serial dilutions. For tests using 96-well plates, human IgG dilutions were prepared in rat serum. The maximum concentration of IgG was 2,875 ng/mL. To show assay performance in 384-well plates human IgG was diluted in PBS with 0.1% BSA. The maximum concentration was 1,000 ng/mL.

Assays using 96 well plates were performed as described in the instructions from Lumigen.⁴

Assays using 384-well plates were performed as described⁴ with the following modifications: antibody mixture (20 µl) was added to wells followed by 10 µl of varying concentrations of IgG for a standard curve. Following incubation 2 µl of background reducing reagent was added.

Plates were read using CLARIOstar test protocols with the following settings which incorporate injection of trigger solution.

Instrument settings

Results & Discussion

The SPARCL assay produces a flash luminescent signal that has a fairly reproducible signature. The signal detected in a 96-well plate using the CLARIOstar corresponds well with what has been previously observed (Figure 2A).

Using MARS data analysis software, a sum of luminescent signals over the 2 second time period of data collection was obtained. From this, we can show that the response is robust with signal to blank calculated to be 587 for the high concentration standard. Furthermore, regression analysis shows that the data fits to a 4-parameter fit as expected (Figure 2B) from which calculation of unknown concentrations could be possible.