Assay Optimization 101: Enhancing Sensitivity, Specificity, and Reproducibility

Assay optimization is central to generating high-quality data in drug development, diagnostics, and molecular biology. The main goal of assay optimization is to increase experiment efficiency, which can be achieved by conserving reagents, minimizing human errors, and ensuring cost-effectiveness. This blog will offer a technical deep dive into best practices for optimizing assay sensitivity, specificity, and reproducibility, specifically in ELISA, PCR, and cell-based assays, highlighting how high-throughput liquid handling automation can be the key to generating accurate and reproducible results. 

Why Assay Optimization Matters

Assay optimization is an essential scientific process where elements of an experimental setup are altered to ensure the most specific, sensitive, and reproducible results. This can involve testing many experimental aspects, including concentrations and volumes of reagents, incubation times, and temperature settings. Low specificity and sensitivity in diagnostic testing can be a major issue, leading to the misdiagnosis of patients, which can negatively affect their outcomes. In R&D, low specificity and sensitivity are equally significant issues that may lead to potential drug candidates being missed or incorrectly identified¹. By optimizing assays, patient diagnostics and R&D can be accelerated and lead to more specific and sensitive results, ultimately reducing time, costs, and potential for human error. 

Key Challenges in Assay Reproducibility and Data Quality 

One main roadblock with assay optimization is manual pipetting, a suboptimal liquid handling technique. Manual pipetting is not only time-consuming and laborious, but it can also introduce the chance of human errors, variability, and waste reagents and precious samples^2,3. This can lead to batch-batch inconsistencies, resulting in unreliable, unreproducible, and poor-quality results, which may need to be repeated, using more time, money, and resources (Fig. 1).  

Assay-Specific Optimization Best Practices  

ELISA: 

• To avoid edge effects, ensure temperature and humidity are consistent across the whole plate and that the same volume is pipetted into each well⁴. 
• Standardizing incubation times and temperatures is essential for improving reproducibility. 
• Using an automated system like I.DOT liquid handler from DISPENDIX will ensure equal and precise volumes are dispensed across all wells, ensuring accuracy and reproducibility. 

Figure 1. Manual pipetting is a suboptimal liquid handling technique that is time-consuming and introduces the risk of errors and contamination. (Source)

PCR: 

• Precision in master mix preparation can be achieved by using clean, non-contaminated equipment⁵.
• Decontamination of work areas and separating pre- and post-amplification areas can reduce the chance of cross-contamination⁶.
• Ensuring low dead volume dispensing using low retention pipette tips and tubes can preserve reagents⁵.
• An automated system like the G.PURE NGS Clean-Up Device from DISPENDIX can optimize reaction setup, preserve reagents and consumables, and ensure accurate and precise delivery.
  
  

Cell-Based Assays: 

• Gentle liquid handling can avoid cell stress and maintain cell viability across the assay⁷.
• Ensure consistent and uniform volumes are dispensed across plates to reduce well-to-well variation⁷.
• Ensure a clean work station and use aseptic techniques to lower the chance of contamination from cells⁷.
• Using an automated system like the non-contact I.DOT liquid handler ensures accurate, gentle dispensing, with equal volumes dispensed into each well while minimizing contamination risks^7,8. 

Role of Liquid Handling Automation in Optimization 

Precision, accuracy, and repeatability are critical in the biopharma industry. Manual workflows are inefficient, they have a high risk for human error, waste reagents and precious samples, and are time-consuming^2,3. Automated workflows can reduce all of these inefficiencies. Automated liquid handling can enable results to be obtained with speed, high-throughput, and traceability, all with minimal errors. This leads to more sensitive, specific, reliable, and reproducible results. 

DISPENDIX Solutions: Empowering Reproducibility Through Technology 

Automated liquid handling systems like the I.DOT liquid handler and G.PURE from DISPENDIX enable user-friendly solutions for assay optimization for many liquid handling workflows. The I.DOT liquid handler provides precise automated non-contact dispensing from picoliter to microliter scale, allowing accurate reagent dispensing while reducing reagent waste (Fig. 2). This system is ideal for miniaturizing PCR setups, reducing assay volumes while maintaining accuracy and achieving cost savings. The I.DOT liquid handler can also facilitate ELISA optimization, capable of dispensing 10 nanolitres across a 96-well plate in just 10 seconds and across a 384-well plate in only 20 seconds, enabling high-throughput workflows that are accurate and reliable.

Figure 2. The I.DOT liquid handler from DISPENDIX is a non-contact automated liquid handling system that provides accurate, reliable, fast, high-throughput dispensing, essential for assay optimization.

The G.PURE from DISPENDIX is designed to automate bead-based clean-ups, which can be one of the most tedious and time-consuming steps in the next-generation sequencing (NGS) library preparation process. Through the G.PURE’s automated system, error-prone manual pipetting can be avoided, enabling fast, reproducible, and cost-effective results. This reduces hands-on time, allowing researchers to dedicate time to other tasks. 

The G.PREP bundle from DISPENDIX includes the I.DOT and G.PURE instruments offering robust solutions for automating NGS workflows. This enables assay minitaturization down to 1/10th reaction volume and scalability, enabling high-throughput, accurate, and reproducible results. 

Realizing Regulatory and Scientific Benefits 

Automation not only supports assay optimization but can also support data integrity and compliance with regulations such as 21 CFR Part 11. Audit logs and barcode tracking simplify workflow traceability and allow for improved reproducibility in peer-reviewed research and good manufacturing practice (GMP) environments.

Conclusion: Build Better Assays With DISPENDIX

Assay optimization plays a crucial role in enhancing workflow efficiency, ensuring results are accurate, reliable, and reproducible. Liquid handling automation stands out as the optimal solution for simplifying assay optimization. With the I.DOT liquid handler and G.PURE NGS Clean-Up Device from DISPENDIX, you can achieve sensitive, specific, and reproducible results with ease and speed. Whether you are optimizing ELISA screening, PCR setup, or cell-based assays, DISPENDIX is here to help you find the best solution that aligns with your assay optimization needs.

Download the I.DOT brochure or the G.PREP brochure, and take the next step in optimizing your workflows!

References

1. Hughes J, Rees S, Kalindjian S, Philpott K. Principles of early drug discovery. Br J Pharmacol. 2011;162(6):1239-1249. doi:10.1111/j.1476-5381.2010.01127.x
2. Guan XL, Chang DPS, Mok ZX, Lee B. Assessing variations in manual pipetting: An under-investigated requirement of good laboratory practice. J Mass Spectrom Adv Clin Lab. 2023;30:25-29. doi:10.1016/j.jmsacl.2023.09.001
3. Lippi G, Lima-Oliveira G, Brocco G, Bassi A, Salvagno GL. Estimating the intra- and inter-individual imprecision of manual pipetting. Clin Chem Lab Med CCLM. 2017;55(7). doi:10.1515/cclm-2016-0810
4. Greene G, Hodous J, Dintzis RZ, Dintzis HM. Modification, optimization and simplification of the spot ELISA technique for the enumeration of cells secreting anti-hapten antibodies. J Immunol Methods. 1990;129(2):187-197. doi:10.1016/0022-1759(90)90438-2
5. Lorenz TC. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. J Vis Exp. 2012;(63):3998. doi:10.3791/3998
6. Czurda S, Smelik S, Preuner-Stix S, Nogueira F, Lion T. Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination. Burnham CAD, ed. J Clin Microbiol. 2016;54(1):148-152. doi:10.1128/JCM.02112-15
7. Annona G, Liberti A, Pollastro C, Spagnuolo A, Sordino P, De Luca P. Reaping the benefits of liquid handlers for high-throughput gene expression profiling in a marine model invertebrate. BMC Biotechnol. 2024;24(1):4. doi:10.1186/s12896-024-00831-y
8. Schober L, Büttner E, Laske C, et al. Cell Dispensing in Low-Volume Range with the Immediate Drop-on-Demand Technology (I-DOT). SLAS Technol. 2015;20(2):154-163. doi:10.1177/2211068214562450