During the validation of a pharmacokinetic (PK) assay for human IgG drug in rat serum, a technique for detecting homogeneous luminescent immunoassays, using the interaction of acridan and HRP in the presence of hydrogen peroxide, was evaluated.
A commercially available SPARCL kit was purchased and an automated robotic pipetting station was utilized for the handling of liquid. The study examined the possibility of an off-the-shelf, affordable solution for automated PK assays for human IgG drugs dosed in preclinical species.
In the execution of the SPARCL commercial kit, the automated robotic pipetting station provided the liquid handling. It was investigated for hours of labor required, as well as for assay performance including: precision, total error, accuracy, minimum required dilution and dynamic range.
SPARCL™ (Spatial Proximity Analyte Reagent Capture Luminescence) technology is a proximity dependent, chemiluminescent method of detection. In a SPARCL assay, a specific antigen/antibody interaction is used to bring a chemiluminescent compound (acridan) into close proximity with an oxidative enzyme (horseradish peroxidase, HRP). Upon addition of a trigger solution that contains hydrogen peroxide, a flash of light is generated. This flash of light is proportional to the quantity of analyte present in the sample. Often, a background reducing agent (BGR) is used to enhance S/N rations. There are no washing steps and no need to remove excess reactants.
HRP and acridan are brought into close proximity by specific antigen and antibody interaction. Adding the hydrogen peroxide based trigger solution causes a flash of light by interacting with HRP and acridan.
The X-axis displays the time, from zero to one second. The Y-axis shows relative light units (RLU). At 0.15 seconds after the addition of the trigger solution, there is a characteristic spike in light. Following this spike, there is a predictable decay.
Figure 1: SPARCL key components.
Figure 2: A Representative SPARCL assay.
Figure 3: A typical SPARCL luminescence profile.
Figure 4: The Andrew pipetting station.
Figure 5: Typical ELISA workflow in blue and typical SPARCL workflow in green.
Materials and Methods
SPARCL PK Kit: Catalog number IGG-SP-20, Life Diagnostics, Inc. (West Chester, Pennsylvania).
Humira: known also as Adalimumab. It is a fully human recombinant immunoglobin, IgG1, and is an anti-TNF monoclonal antibody. This was obtained from a local pharmacy.
Andrew 1000G liquid handling robot: Andrew Alliance (Boston, Mas- sachusetts)
Pipets: Gilson, Pipetman (Middleton, Wisconsin)
Matrix: Rat serum. Bioreclamation (Baltimore, Maryland). Used at 10% in assay buffer. Life Diagnostics provided the assay buffer.
Luminometer: BMG LabTech’s LUMIstar Omega
The SPARCL PK kit was run, according to directions provided by the manufacturer (Life Diagnostics), by giving the Andrew robot the pipetting instructions. Andrew made the dilutions for the standard curve and QC’s, and pipetted the calibrators, antibodies and QC’s into the wells of the 96 well plates.
The 96 well plate was incubated for 30 minutes at room temperature. This was done on an orbital shaker which was set at 350 revolutions per minute. The plate was moved to the LUMIstar Omega luminometer after the incubation was complete. Instructions were given to the LUMIstar to inject the trigger solution and collect the light emitted.
Table 1: Typical standard curve data. RLU is Relative Light Units.
Table 2: Quality Control samples, high (20 ng/mL) and low (5 ng/mL). Concentration values interpolated from the standard curve are reported.
Conclusions and Future Direction
- The Andrew robot and the Life Diagnostics SPARCL PK kit reduces human error and input, offering improvement in workflow and efficiencies.
- Using the Andrew robot with the Life Diagnostics SPARCL PK kit provides an assay which is precise and accurate for Humira in rat matrix.
- The Andrew system provides compliant software which might make the system useful in regulated laboratories.
- Combining the Andrew robot with the Life Diagnostics SPARCL PK kit might be a way for CRO’s to improve efficiencies, reduce labor, reduce PK assay development cost and time and increase profitability.
- Combining the Andrew robot with the Life Diagnostics SPARCL kit might provide a solution for labs that measure IgG drug concentration in a preclinical setting.
About Andrew Alliance S.A.
Andrew Alliance is an independent, privately financed company, based in Geneva, Boston and Paris. The company was created in March 2011.
Andrew Alliance is dedicated to advance science by working with scientists to create a new class of easy-to-use robots and connected devices that take repeatability, performance, and efficiency of laboratory experiments to the level required by 21st-century biology.
Start with meeting customer needs, end with customer feedback.
Andrew Alliance delivers solutions that are focused on customer needs, both today and in the future. Our products are manufactured to the highest standards, using a range of carefully selected, proven, and sustainable technologies, that ensure both high performance and reliability. We actively seek continuous customer feedback, in order to guarantee the best possible design outcomes.
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