Optimizing a Corning Epic Biochemical Assay An Example with the Lactate Dehydrogenase A
Optimizing a Corning® Epic® Biochemical Assay: An Example with the Lactate Dehydrogenase A
Authored by: Nicolas Andre, Ph.D., Sylvie Bailly Corning Incorporated Life Sciences, 7 bis Avenue de Valvins Avon Cedex, France
Originally published in SnAPPShots: A brief technical report from the Corning Applications Group. Reprinted with permission.
Assay development is a crucial step towards biochemical investigation and in drug discovery process. The Epic System, with its 384 well microplate and HTS-compatible reader, allows testing a large number of conditions in a limited time, and leads to reduced assay development time, decreasing work load and cost.
The following demonstrates the steps for optimization of an assay developed for the well-characterized lactate dehydrogenase A (37, 380 Da, pI = 8.38), a key enzyme involved in the last steps of anaerobic glycolysis. Furthermore, this activity level serves as a marker in opportunistic infections associated with HIV . To assess protein binding, we used its natural ligand, nicotinamine adenine dinucleotide (NADH, 663 Da).
Materials and Methods
Epic biochemical assays are performed on amine-coupling surfaces (Corning Cat. No. 5041 and 5046XX1). In this study, Epic microplates (Corning Cat. No. 5046XX1) were activated with 10 μL of 200 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 50 mM sulfo-N-Hydroxysulfosuccinimide (sNHS) (Pierce Cat. No. 22981 and 24510), and incubated for 30 minutes at room temperature. The microplates were subsequently washed 3 times with 20 μL H2O with 384 well pipettor (CyBi®-Well).
Protein immobilization was accomplished by adding 10 μL of LDHA protein (provided by AstraZeneca, UK) at the specified concentration in the buffer listed below (Table 1) using a 384 well pipettor (CyBi-Well), and incubating overnight at 4°C. The microplates were subsequently blocked with 150 mM borate buffer (pH 9.2, 200 mM ethanolamine) for ten minutes, and washed three times with assay buffer (50 mM Hepes pH 7, 300 mM NaCl, 1.5 mM MgCl2, 1 mM DTT), and finally 15 μL assay buffer was added.
|20 mM sodium citrate||4.5|
|20 mM sodium citrate||5.0|
|20 mM sodium acetate||5.5|
|20 mM sodium phosphate||6.5|
|20 mM sodium phosphate||7.0|
|20 mM sodium phosphate||7.5|
The binding assay was run on a Corning® Epic® instrument. After 25 minutes thermal equilibration, a baseline read was taken on the Epic microplates with protein immobilized in assay buffer. In the next step, 15 μL of compounds were added to the microplates with a 384 well pipettor (CyBi®-Well), and then mixed 10 times. The final read was taken after 25 minutes thermal equilibration.
pH Scouting: In order to identify the best conditions for protein immobilization, a pH scouting analysis was performed using six different buffer solutions (pH values ranging from 4.5 to 7.5), and 50 μg/mL LDHA as the standard starting concentration (Fig. 1). The maximum immobilization level was obtained in 20 mM sodium acetate buffer (pH 5.5) with more than 2,000 pm.
Nevertheless, the maximum binding signal with 50 μM NADH was obtained when protein was immobilized at pH 7.5 (~15 pm). Therefore, the maximum immobilization level did not necessarily lead to the highest binding signal. The protein activity might be decreased when incubated at pH values lower than 6.5.
LDHA concentration optimization: Once the immobilization buffer was selected, different LDHA concentrations were tested ranging from 30 to 100 μg/mL for immobilization. The immobilization level observed remained constant from 40 to 100 μg/mL at ~2,000 pm, and started to decrease at 30 μg/mL (Fig. 2).
In order to obtain binding statistics and determine the affinity of NADH to LDHA, a dose response binding assay was performed on three different concentrations of immobilized protein (Fig. 3). NADH binding to LDHA was saturable and specific at all three concentrations tested. The affinity measured, ~12 μM, was in the range expected (5 μM) , and the maximum binding signal was ~15 pm.
The robustness of the assay was determined in the presence of 5% DMSO, the conditions required for high concentration ligand screening. To improve robustness, we also tested for the presence of Tween® 20 (0.01%).
Three microplates were tested in parallel with 64 wells per condition to assess the reproducibility of the assay. These results are summarized in Table 2.
In presence of DMSO, the ligand binding level reached 18 pm with low variability over the three microplates (less than 10%). The assay robustness (Z') was improved in the presence of 0.01% Tween 20 and was >0.5 for the three microplates.
|Figure 1. pH Scouting. Immobilization levels were monitored using different buffers with pH values ranging from 4.5 to 7.5 (50 μg/mL). The binding levels of 50 μM NADH is also monitored to ensure protein functionality.|
|Figure 2. LDHA Concentration Optimization. Immobilization levels are monitored using different protein concentrations (30, 40, 50 and 100 μg/mL) in sodium phosphate buffer (pH 7.5).|
|Figure 3. NADH Dose Response Curve. Specific binding of NADH to LDHA is monitored for protein immobilized at different concentrations (30, 40 and 50 μg/mL). The binding of a non-specific compound (red) to LDHA (50 μg/mL) is also monitored.|
|3 times N = 64||Response (pm) at 100 μM NADH||CV (%)||Z'|
|No Tween® 20||18.9||9.0||0.4 ±0.13|
|0.01% Tween 20||18.6||9.7||0.55 ±0.3|
- The Corning® Epic® System enables label-free direct biochemical assay for small molecule binding to an immobilized protein. Several simple steps can be performed and are necessary to optimize the assay, including selecting the right pH and buffer conditions, optimizing concentration of target bound, and evaluating assay robustness with respect to buffer conditions.
- Applying the general strategy described here for most targets investigated can minimize the cost and time required for assay development.
- LDHA binding to NADH, evaluated using the Epic System after optimization of assay parameters, demonstrates high specificity to its natural ligand.
- Furthermore, the Z' was robust (>0.5), indicating that this assay is suitable for up-scaling for screening.
- Combining the high sensitivity and throughput, the Epic System is a reliable tool to be used in HTS biochemical assay for the drug discovery process.
- ↑ Boldt MJ and Rai TR. Utility of lactate dehydrogenase vs. radiographic severity in the differential diagnosis of pneumocystis carinii pneumonia. CHEST 1997; 111:1187-1192.
- ↑ Srivastava DK, Smolen P, Betts GF, Fukushima T, Spivey HO and Bernhard SA. Direct transfer of NADH between α-glycerol phosphate dehydrogenase and lactate dehydrogenase: Fact or misinterpretation? Proc. Natl. Acad. Sci. USA 1989; 86:6464-6468.
|Click [+] for other articles on||The Market Place for Lab Automation & Screening||Analytical Equipment, Measuring, Testing Detection Devices|