Application Note 18「Best practices for performing quantitation assays using the BLItz System」

APPLICATION NOTE 18 Best practices for performing quantitation assays using the BLItz System Yixin Lin and Pui Seto, Technical Support Scientists, ForteBio LLC Introduction 3 Unused biosensors must be stored inside the original biosen- sor pouch and sealed with the desiccant pack, and must stay The BLItz® system provides a simple, rapid Dip and Read™ dry. The sealed biosensors should then be stored at room approach for protein quantitation in a small, affordably priced temperature in a dry location. Biosensors cannot be frozen. If personal assay system. With BLItz, proteins and antibodies can a biosensor is ever stored frozen, it loses its activity and can- be quantitated in a matter of seconds with high specificity and not be used any more. sensitivity, even in crude samples. The system utilizes the same proprietary Bio-Layer Interferometry (BLI) technology as ForteBio’s 4 Turn on the BLItz system and allow it to warm up for at least Octet platform, enabling real-time analysis of interactions on the one hour prior to use. surface of disposable fiber optic biosensors. Affinity, concentration 5 Take note of the temperature of the room, the biosensor and binding kinetics can be measured right at the bench in a 4 μL lot number, and date to compare data run across multiple drop of sample. days or weeks. Traditional techniques for determining concentration of a target 6 Ensure that the drop holder is clean before use. protein such as ELISA and HPLC are both elaborate and time consuming, especially when analyzing complex matrices. For a If the drop holder is still wet, use a Kimwipe® to clean it. bioprocess development and production applications, the NOTE: To prevent cross contamination, do not reuse the use of more rapid protein analysis techniques enables timely, same part of the Kimwipe to perform another cleaning step. informed process decisions. The BLItz system can both stream- line workflows and obtain rapid, direct quantitation of proteins i Add 10–20 µL of sample diluent directly into the drop in crude matrices. holder. Do not let the liquid overflow. Pipette up and down five times, then dab off the excess liquid using a In this application note, we’ll describe best practices for get- Kimwipe. Finally, dry the inside of the drop holder very ting started with the BLItz system, as well as developing and well with a corner of the Kimwipe using your thumb running quantitation assays, sample and biosensor prepara- nail to push the Kimwipe into the drop holder and turn tion, and data analysis. clockwise a few times. Getting started ii Repeat the above cleaning step a total of 3–5 times. 1 Place the BLItz system in a room with a constant temperature iii Inspect the dryness of the drop holder under light to away from AC vents, air fans, heat-generating equipment, and ensure it is completely dry. direct sunlight. Do not place the system on the same bench b If samples are allowed to dry out in the drop holder, a with a centrifuge. solution of 0.5 M HCl can be used to clean it. 2 When using the BLItz system, all reagents, including the i Add 10–20 µl of the HCl solution directly into the hydration buffer, should be brought to room temperature prior drop holder. Rub the drop holder with a swab, and to the start of an experiment. then dab off the liquid with a Kimwipe. If excess HCl overflows from the drop holder, wipe off the excess liquid with a Kimwipe. 1

ii Place the drop holder in a 50 mL conical tube with 6 If the concentration of an unknown is too high (outside the 30–50 mL of purified water. Shake the tube back and linear range of the standard curve), dilute the unknown into forth 5–10 times and discard the used water. Repeat the linear range and repeat the experiment (Figure 1). the wash a total of 3 times. 7 Test assay accuracy by measuring controls, i.e. samples iii Next rinse the drop holder with sample diluent, using where known amounts of purified analyte are spiked into the the steps listed in step a. Do this a total of 5 times. desired matrix. The recovery values should fall within an ac- ceptable range (± 20%). c To confirm cleaning, you can run a Quick Yes/No experi- ment to verify that no contaminating proteins have been 8 Test assay precision by measuring replicates of the same left behind to bind to a biosensor. sample and calculating the coefficient of variance (CV). Two or three replicates can be used depending on the assay Considerations for developing a successful criteria defined by the user. quantitation assay Samples and biosensor preparation 1 Standards and unknowns should be of the same protein. 1 Hydrate biosensors for at least 10 minutes prior to use. Bio- 2 All samples measured for quantitation (including standards sensors should be hydrated in a biosensor tray assembly with and unknown samples) should use exactly the same matrix. a hydration plate underneath which has at least 200 µL of For example, if the standards in buffer 1 are diluted 10x in hydration buffer at corresponding locations. The buffer used buffer 2, unknowns in buffer 1 should also be diluted 10x in for hydration should be identical to the buffer of the samples buffer 2. This ensures that the final concentration of the bulk to be measured. components in the final matrix of standards and unknowns should be the same. 2 Hydration buffer should ideally be fresh for every biosensor that needs to be hydrated. Do not reuse the hydration buffer 3 For the first run, always measure a reference biosensor us- to hydrate additional biosensors. ing buffer only to identify any non-specific binding, matrix or background issues. If a high background signal is observed, 3 Once biosensors are wet, they must stay wet and should not the assay should be optimized. be allowed to dry. They may be stored at 4°C, with the tips of the biosensors submerged in hydration buffer for up to one 4 Use at least 6–8 known sample concentrations to create a day, with the understanding that the storage time is deter- standard curve. mined by the stability of the proteins on biosensors which 5 Samples measured should be within the concentration range should be validated by the user. If the biosensors dry out, defined by the standard curve, and within the linear dynamic they lose activity and cannot be used any more. range of the standard curve (Figure 1). LLOQ Linear Dynamic Range Poor Absolute Quantitation ULOQ Poor Absolute Quantitation Concentration (µg/mL) Figure 1: Standard curve showing limits of detection and quantitation. For optimal results, always work within the linear dynamic range of the standard curve. 2 Binding Rate

4 Always prepare all samples including dilutions before start- 9 A new standard curve should be created if the lamp has ing a BLItz measurement. Use gentle tapping or pipetting up been changed and calibrated. Samples measured after a and down to mix samples. Do not vortex samples to avoid lamp change should use this new standard curve to calcu- formation of bubbles. If bubbles form, perform a quick spin late concentrations. to remove them. Data analysis Running the assay During data analysis: 1 The drop holder should be cleaned thoroughly and dried completely before use. Always clean the drop holder imme- • Define the biosensor with the buffer blank measurement as a diately after use, do not let it dry before cleaning. For details reference by checking the Ref. box. on the cleaning procedure, please refer to the Getting started • Subtract the reference biosensor. section, step 6. • For standard curve application, always first apply the linear-point-to-point function as a curve fitting function and 2 During measurement, follow the on-screen prompts to observe the binding trend of the data. Then apply 5PL or enter assay settings (experiment settings, run settings, linear fits and look for the residual of the curve fitting. If 5PL and sample ID), open the cover, pipette samples into the or linear fits do not perform well, use the linear-point-to- drop holder, mount the biosensor, and close the cover as point curve function. Exclude outlier data points if they skew instructed. Always pipette samples into the drop holder the curve fitting. before mounting a biosensor. The time between mounting a biosensor to measurement should be as short as possible, • Ideally, only include data points with calculated binding rate ideally within 15 seconds. values less than 2. This is a user-defined value, but our gen- eral recommendation is 2 or less. 3 Use the Create Standard Curve function to create standard • Inspect the equation, equation parameters, R2 and Chi2 of the curves and the Quantitate Sample function to quantitate standard curve. Choose a standard curve with R2 greater than unknowns. 0.95 and Chi2 less than 3. 4 The drop holder should be used for quantitation assays. • Inspect the locations of unknown samples on the standard Pipette 4 µL of sample into the drop holder. Avoid introducing curve. If they are outside the concentration range defined air bubbles when pipetting. by the standards, the results of these unknowns are not reli- 5 Multiple drop holders can be used in a staggered fashion to able and should be excluded. If the unknowns are located improve workflow. on the upper end of the standard curve that is plateauing out of the curvature (outside the linear dynamic range), 6 Once used, a biosensor should be discarded or regener- the results are not ideal for accurate quantitation. Sample ated. A spent biosensor cannot be reused without proper measurement should be repeated by diluting the samples regeneration. so the concentrations fall within the linear dynamic range of 7 When saving standard curves to reuse for other unknowns at the standard curve (step Figure 1). a later time, always note the standard name, matrix, specific • Copy and paste data from the table into an Excel file as ambient temperature at the time, shaking speed, and date to needed for any additional calculations. compare curve data across multiple days or weeks. • Click on Create Report to save report as a PDF file. 8 Only load a standard curve that has matched matrix, matched protein, matched shake speed, and matched temperature for calculating the concentration of unknowns. ForteBio ForteBio Analytics (Shanghai) Co., Ltd. Molecular Devices (UK) Ltd. Molecular Devices (Germany) GmbH 47661 Fremont Boulevard No. 88 Shang Ke Road 660-665 Eskdale Bismarckring 39 Fremont, CA 94538 Zhangjiang Hi-tech Park Winnersh Triangle 88400 Biberach an der Riss 888.OCTET-75 or 650.322.1360 Shanghai, China 201210 Wokingham, Berkshire Germany www.fortebio.com fortebio.info@moldev.com RG41 5TS, United Kingdom + 00800 665 32860 +44 118 944 8000 uk@moldev.com ©2019 Molecular Devices, LLC. All trademarks used herein are the property of Molecular Devices, LLC. Specifications subject to change without notice. Patents: www.moleculardevices.com/product patents. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. AN-4018 Rev C