A poster presented at Lab Automation discussing methods used to evaluate the sensitivity, reproucibility and variance on various readers.Read less
![Techniques For Evaluating Detectors in High Throughput Screening Susan Foltin, Pfizer Global Research and Development, 2800 Plymouth Rd, Ann Arbor, MI 48105 ABSTRACT In many cases, the rate-limiting step in a fully automated high throughput screening system is the time required to read a plate. This is especially true for plate densities of 384 or higher. The addition of a second detector on most systems does not double the throughput, as plate transport becomes the rate-limiting step. Therefore, detectors that can read plates faster without sacrificing sensitivity or functionality are desirable for our systems. We have generated a series of experiments to evaluate new detectors against the detectors that are currently used in our screening system. We describe these experiments and how we interpret the results in deciding whether or not a new detector meets our lab's guidelines for sensitivity, precision and speed. INTRODUCTION In HTS assays, the ability to read high density plates, such as 384 or 1536 formats, has become one of the primary rate-limiting steps in achieving greater speed without loss of sensitivity or accuracy. This can be alleviated by configuring the screening system with two detectors which can be pooled for data collection. However, increasing the number of detectors on a robotic system containing a single articulated arm does not directly increase an assay’s throughput proportionally, as the transportation becomes rate-limiting. Therefore, a single detector that can read high density plates more rapidly while maintaining the sensitivity and function may be more desirable. We have developed a systematic approach to evaluating and comparing detectors for use in HTS. When evaluating detectors, there are several key factors or characteristics that we use to assess whether the addition of this instrument will be beneficial to achieving our goal of executing a high throughput screen. These factors include: Initially detector evaluations are carried out through discussion with the vendor and or through literature. Detectors of interest are subjected to a systematic performance evaluation in our lab in comparison with those detectors currently in use in our laboratory. This poster will focus on how each component is individually tested , evaluated and compared. ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],MATERIALS AND METHODS The detector used as the standard for comparison in our laboratory will be referred to as Reader A, and the test instrument as Reader B . Both detectors have the ability to perform absorbance, fluorescence and fluorescence polarization detection. Absorbance All absorbance studies were performed using 2.7 mM tartrazine ( Sigma, catalog # T-0388 ) made in distilled water and filtered. All dilutions were performed in distilled water and read at 405 nm. Black, clear bottom 384 well Falcon plates ( catalog # 35-3962 ) were used for all measurements. Detectors were evaluated for lower limit detection as well as variance in absorbance across a 384 well plate. For variance analysis, 50 µ L of a 200 µ M Tartrazine solution was titrated across a black, clear bottom 384 well Falcon plate using a calibrated Matrix pipettor and the absorbance was read at 405 nm using 10 (ten) and 1 (one) flash setting. The average optical density and standard deviation across all wells was calculated using Excel. Coefficient of variance values (%CV) are determined by dividing the standard deviation by the average. The standard criteria for variance across all wells on a plate in our laboratory is a value of 5% or less. The recorded values for read time do not reflect time taken to load / unload plate (considered to be relatively the same for most readers). For sensitivity (lower limits) analysis, a stock solution of tartrazine is diluted to 450 µM, 50 mL final volume, in distilled water. Serial dilutions of 1:1 in 50 mL are prepared. Using a calibrated Matrix pipettor, 50 µL of each dilution is added to all 24 wells of a single row on a 384 well black, clear bottom Falcon plate, with the last row containing 0 µM for background. Data is collected by both Reader A and B. Optical density is plotted against concentration and a linear regression is performed by Excel. The lower limit detection (sensitivity) is assessed by determining the point where the window between signal and background is nearing 3 standard deviations of the background ( ratio above background ): Fluorescence Fluorescence sensitivity studies were performed using 5-(&6)-carboxyfluorescein (mixed isomers, Molecular Probes, catalog # C-194 ) (CF). A stock solution of 10 mM CF was prepared in 100% DMSO ( Mallinckrodt, catalog # 4948 ) with subsequent dilutions made in 10 mM Na Borate buffer, pH 9.2 ( VWR, catalog # VW3969-2 ). Detectors were set to excitation at 485 nm and the emission at 535 nm for measuring CF concentrations. To determine the lower limits for fluorescence measurements, a stock solution of CF is diluted to 10 µM, 50 mL final volume, in 10 mM Na Borate, pH 9.2. Serial dilutions of 1:1 in 50 mL were prepared in 10 mM Na Borate, pH 9.2. Using a calibrated Matrix pipettor, 50 µL of each dilution is added to all 16 wells of a single column on a 384 well solid black Costar plate, with the last row containing 0 µM for background. Data is collected by both Reader A and B. Relative fluorescence units (RFUs) are plotted against concentration and a linear regression is performed by Excel. The lower limit detection (sensitivity) is assessed by the same equation used for absorbance. RESULTS Figure 1. Reader B variance in absorbance across entire plate at different flash settings. Figure 2. Reader A absorbance linear range, scatter and lower limits at different flash settings. Figure 3. Reader B absorbance linear range, scatter and lower limits at different flash settings. Figure 4. Reader A vs. B comparison for fluorescence linear range, scatter and lower limits (at 10 flashes). Figure 5. Reader A vs. B comparison for fluorescence polarization using a standard calibration curve. DISCUSSION ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],CONCLUSION Reader B’s performance maintains the sensitivity needed to detect the standard assays currently being conducted in our laboratory, while allowing for a higher throughput due to faster read times. The cost to purchase a single unit with all the desired filters and mirrors is slightly higher than Reader A and less than other readers in the lab. Therefore, we would consider using Reader B as a stand-alone unit and as an integrated reader in our automated HTS system once the robotic integration issues are resolved. Fluorescence Polarization For ease of measuring fluorescence polarization, an 12-point calibration curve was generated using the standard provided in the TKXtra FP kit ( Molecular Devices, catalog # 44-0009 ). The reported literature value for point of inflection is 20.4 nM Calibrator. All fluorescence measurements were made using 485 nm excitation and 535 nm emission. For both fluorescence and fluorescence polarization, solid black, 384 well Costar plates ( catalog # 3710 ) were used. Titration of the Calibrator that comes as a standard with Molecular Devices’ TKXtra is prepared according to the kit’s instructions. The titration ranged from 0 – 250 nM with serial dilutions of 1:2. The literature value for the point of inflection is 20.4 nM. A calibrated Matrix pipettor is used for all additions into a 384 well solid black Costar plates. Data was collected by both Reader A and B at 485 nm excitation and 535 nm emission to measure light in both the parallel and perpendicular planes. Milli P values were calculated based on the equation: (parallel – perpendicular)/(parallel + perpendicular)*1000. Point of inflection was determined using a general sigmoidal curve (with a Hill slope, a to d) of concentration plotted against corresponding milli P values.](https://image.slidesharecdn.com/pfizerposterpdf-12665487725116-phpapp01/85/Techniques-for-Evaluating-Detectors-in-High-Throughput-Screening-1-320.jpg)
