(M1230-09-49) Combining Acoustic Liquid Handling with the Gyros Immunoassay Platform for Bioanalysis of Therapeutic Antibodies in Small Volume Samples

Purpose: One of the practical difficulties that may be encountered by bioanalytical scientists is the limited amount of sample obtainable from animals at each time point. The sample volume may not be sufficient to run multiple tests. For certain studies, for instance in transgenic mice, the matrix volume is severely limited. In the current work, we explored the feasibility of acoustic liquid handling (ALH) instrumentation for preparation of standards, quality controls (QC) and sample dilutions for analysis on the Gyros microfluidic immunoassay platform.

Methods: We previously developed a pharmacokinetic (PK) immunoassay for a preclinical study to quantify a therapeutic antibody (TAb) in mouse serum samples on the Gyros platform. Biotinylated target protein was used as a capture reagent and AlexaFluor647-labeled mouse anti-human IgG (Fc specific) monoclonal Ab was used for detection. The minimum required dilution (MRD) for this assay was 10-fold in MRD diluent, containing PBS, 1% BSA, 0.05% Tween 20. In 100% mouse serum, the quantitative range of the assay was from 20 to 5000 ng/mL, Using the PK assay and study samples, we evaluated the Labcyte ECHO 525 ALH to make a set of standards/QC and sample dilutions via direct dilution which mirrored the manually generated standards/QC and sample dilutions.

Results: No significant difference was observed between the ECHO-prepared and manually prepared standard curves, indicating that ALH instrumentation can be used for preparation of standards and QC. The assay had acceptable precision and accuracy for both sets of standards and QC: CV for standards was below 20% and QC recovery was within +/- 20% of nominal values. To demonstrate that acoustic liquid handling instrumentation can be used for sample pre-dilution and preparation of MRD, 4 replicates of 10 study samples were prepared using ECHO and analyzed on the Gyrolab. The results showed excellent within assay performance of automatically prepared samples, all samples had acceptable CV percentage (below 20 %), moreover, for 9 out of 10 samples, CV% was < 10% (Table). Excellent agreement was observed between the original study results and the data generated from ECHO-prepared samples (r=0.99, p< 0.0001).

Conclusion: The results suggest that 10 microliter sample volume allows for single or multiplexed bioanalysis. Our work presents a novel application of acoustic liquid handling leading to the successful integration of such with the Gyros microfluidic immunoassay platform for bioanalysis.

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