Fully-Automated Workstation for Direct camp Enzyme Immunoassay Kit Application Note 233 Nikki Popp, David King and Joan Stevens, Ph.D., Gilson, Inc. Introduction Adenosine 3, 5 -cyclic monophosphate (cyclic AMP; camp) is one of the most important second messengers involved as a modulator of physiological processes. camp is also involved in regulating neuronal, glandular, cardiovascular, immune and other functions and actions. A number of hormones are known to activate camp through the action of the enzyme adenylate cyclase which converts ATP to camp. These hormones include a variety of anterior pituitary peptide hormones such as corticotropin (ACTH), glucagon, calcitonin, thyroid stimulating hormone (TSH) and luteinizing hormone (LH). camp has been shown to be involved in the cardiovascular system, nervous system, and immune mechanisms, cell growth and differentiation and general metabolism. There remains considerable interest in the measurement of intracellular camp in tissues and cells cultures, and automating the process may help to provide an understanding of the physiology and pathology of many disease states. Figure 1. Chemical formula for cyclic AMP. The 940 Workstation is a multiple-function X/Y/Z robot that allows immediate access to multiple tools on the same head resulting in a time savings and increased efficiency. The head consists of 1-, 8- and 96-channel pipettors and a gripper, and has the ability to utilize either disposable pipette tips or fixed probes. It is capable of integrating third-party accessories and devices into the system and has a flexible, customizable deck design which accommodates virtually any liquid handling or sampling November 2004 Page 1 319333-01
protocol ranging from PCR preparation and cleanup, protein purification, nucleotide labeling and cherry picking to high-throughput screening and protein crystallography. The purpose of this experiment is to automate an EIA Direct cyclic kit via a 940 Workstation. In its purest sense, automation defines a system that completes an assay or multiple tasks without any operator intervention. In order to be able to complete an ELISA from start to finish, it is required to integrate peripheral or third-party equipment into the system. Integration of peripheral equipment requires the automated system to have the ability to talk to the equipment. On the surface, this point may seem insignificant; however, accomplishing a symbiotic interface between peripheral equipment and the automated instrument and controlling software can be quite a challenge. The 940 Workstation offers a novel approach to interfacing peripheral devices via run tasks that employ executable files. The employment of executable files for the individual peripheral instruments offers complete control and interactive responses from the integrated equipment. The information presented in this application will demonstrate the power and usefulness of this concept for an enzyme immunoassay kit currently employed in high-throughput biological laboratories. Photo1. Gilson 940 Workstation. Materials & Methods 940 Workstation Hardware Characteristics Stationary deck X/Y/Z robot holds up to five tools (1-, 8- or 96-channel pipettors and gripper) which allows immediate access to multiple tools on the same head saving time and increasing efficiency Single-channel pipettor accesses individual wells for cherry picking applications Flexible, customizable deck design accommodates 200+ microplates of virtually any liquid handling or sampling protocol Ability to process vials and microplates on the same deck Utilizes disposable pipette tips and/or fixed probes Ability to integrate third party accessories and devices into the system (This application utilizes a H&P VarioMac teleshaker, a Molecular Devices Vmax plate reader and a Bio-Tek ELx405 plate washer.) November 2004 Page 2 319333-01
1 2 3 4 Photo 2. Illustration of robot head: Tool 1, 96-channel pipettor; Tool 2, 1-channel pipettor; Tool 3, gripper; Tool 4, 8-channel pipettor. Photo 3. 96-channel pipettor with 50-µL disposable tips. 940 Workstation Control: 745 Control Software Rack Layout Screen Contains multiple, unique layers which allow various plates to be accessed at a particular location Quick and easy optimization by double clicking on element of interest Screen 1. 745 Control Software Rack Layout screen. November 2004 Page 3 319333-01
Deck Configuration Screen Simulation mode allows user to view the assay prior to running Screen 2. 745 Control Software Deck Configuration screen. Method Development Screen Drag and drop tasks allow quick and easy method development and optimization changes Screen appears as a full grid allowing the user to view each value for all steps Screen 3. 745 Control Software Method Development Screen. The tasks are accessible by a simple double-click. Tasks that are highlighted are required; those that appear gray are optional. November 2004 Page 4 319333-01
Description of the Procedure Aspirate neutralizing reagent from source plate using 8-channel tool. Dispense reagent into LabSystems Immunoassay nonspecific binding (NSB) wells; dispose of tips. (Repeat for zero standard (B 0 ) and all standards and horse serum samples.) Aspirate 0.1M HCl from source plate using 8-channel tool. Dispense HCl into NSB wells; dispose of tips. (Repeat for B 0.) Aspirate standard and/or horse serum sample from source plate using 8-channel tool. (Different concentrations of camp are spiked into the second and third samples, resulting in three unique samples.) Dispense standards and/or horse serum samples into ELISA plate; dispose of tips. Aspirate blue camp conjugate from source plate using 8-channel tool. Dispense conjugate into NSB wells; dispose of tips. (Repeat for B 0 and all standards and horse serum samples.) Aspirate yellow camp antibody from source plate using 8-channel tool. Dispense antibody into B 0 wells, dispose of tips. (Repeat for all standards and samples.) Use gripper to move ELISA plate to shaker location. Shake for two hours at 500 rpm. Move plate to washer position. Wash plate three times with wash solution. Move plate back to target location. Aspirate blue camp conjugate from source plate using 8-channel tool. Dispense conjugate into total activity (TA) wells; dispose of tips. Aspirate p-npp substrate from reservoir using 96-channel tool. Dispense substrate into all wells of the ELISA plate and incubate at room temperature for one hour. Aspirate stop solution from reservoir using 96-channel tool. Dispense stop solution into all wells of the ELISA plate. Use gripper to move ELISA plate to reader position. Read plate at 405nm; manually subtract the mean optical density of the blank wells from all readings. Standard Volume of 0.1M HCl (µl) Volume Added (µl) Table 1. Dilution table for making standards 1 5 from 2,000 pmol/ml camp stock. camp Concentration (pmol/ml) 1 900 100, stock std. 200 2 750 250, std. 1 50 3 750 250, std. 2 12.5 4 750 250, std. 3 3.12 5 750 250, std. 4 0.78 November 2004 Page 5 319333-01
Blank Total Activity (TA) Non-specific Binding (NSB) Zero Std. (B 0 ) Standards Samples Well ID* 1 8 9 16 17 24 25 32 33 72 73 96 Neutralizing Agent 50 µl 50 µl 50 µl 50 µl 0.1M HCl 150 µl 100 µl Std. and/or Sample 100 µl 100 µl Conjugate 50 µl 50 µl 50 µl 50 µl Antibody 50 µl 50 µl 50 µl Incubate 2 hrs, shake Yes Yes Yes Yes Yes Yes Wash 3x @ 200 µl Yes Yes Yes Yes Yes Yes Conjugate 5 µl Substrate 200 µl 200 µl 200 µl 200 µl 200 µl 200 µl Incubate 1 hr @ RT Yes Yes Yes Yes Yes Yes Stop Solution 50 µl 50 µl 50 µl 50 µl 50 µl 50 µl Table 2. Assay protocol flow chart. *Microplate well IDs are numbered back-to-front and left-to-right. Results Calculations To calculate the average Net Optical Density (OD) bound for each standard and sample, subtract the avg. NSB OD from the avg. bound OD: Avg. Net OD = Avg. Bound OD Avg. NSB OD To calculate the binding of each pair of standard wells as a percentage of the zero standard (B 0 ): Percent Bound = (Avg. Net OD Net B 0 OD) x 100 After these calculations were performed, the percent bound was plotted against the concentration of camp for the five standards. A straight line was approximated through the points and the unknowns concentration of camp was determined by interpolation. (See Table 3 and Graph 1.) Avg. Net OD* % Bound camp (pmol/ml) Blank 0.0810 Non-specific Binding Wells (NSB) 0.0002 0.06 Zero Std. (B 0 ) 0.3430 100.00 0 Std. 1 0.0180 5.25 200 Std. 2 0.0460 13.41 50 Std. 3 0.1070 31.20 12.5 Std. 4 0.2110 61.52 3.125 Std. 5 0.2860 83.38 0.781 Sample 1 0.0820 16.80 28.89 Sample 2 0.1600 31.06 8.63 Sample 3 0.2210 62.55 2.18 Table 3. Results of automated camp Immunoassay Kit. *Average net ODs are reported as the average sample reading of all eight wells. November 2004 Page 6 319333-01
camp Immunoassay Ki % Bound 120 100 80 60 40 20 0 y = 92.993x -0.5088 R 2 = 0.9698 0 50 100 150 200 250 camp conc. (pmol/ml) Graph 1. Power curve results of five standards. Conclusions The results of the experiment demonstrate that the 940 Workstation is an effective liquid handling instrument for automating enzyme immunoassays. The Workstation was able to integrate a H&P VarioMac teleshaker, a Bio-Tek plate washer and a Molecular Devices Vmax plate reader into the deck design which allowed for the EIA Direct Cyclic Kit to be fully automated without any operator intervention. These results, coupled with hardware characteristics that include a multiple-tool head, the ability to utilize either disposable pipette tips and/or fixed probes, a flexible, customizable deck design and the ability to process vials and microplates on the same deck and integrate other devices into the system, make the 940 Workstation a logical choice for applications ranging from protein crystallography to PCR preparation and cleanup, protein purification, nucleotide labeling and ELISAs. Gilson, Inc. World Headquarters Middleton, WI 53562 USA Telephone: 800-445-7661 or 608-836-1551 Fax: 608-831-4451 www.gilson.com Gilson S.A.S. 19, avenue des Entrepreneurs, F-95400 VILLIERS LE BEL France sales@gilson.com, service@gilson.com, training@gilson.com November 2004 Page 7 319333-01