A. Agar-Diffusion Microbial Assays
Liquid or cell-free assays have become very popular in the pharmaceutical arena for the purpose of drug discovery, particularly due to their adaptability to automation, miniaturization, and hands-off data collection and management. However, microbial-based agar diffusion assays provide a great deal of information about a sample that is not possible to obtain with liquid-type assays. The effect of a large range of concentrations of a compound on the test organism can be assessed with a single application, and assay sensitivities in the low nanogram range can be obtained with good reproducibility. In addition, sample activity can be evaluated despite the presence of toxic effects that can mask potential activity when conducted in a liquid assay. Further, contamination from any interfering organism(s) can be readily detected and scoring judgments made accordingly. A disadvantage associated with agar diffusion assays is the need to collect and analyze data manually unless some form of sophisticated image analysis is available. Thus data management for agar-based assays is not as efficient as with liquid-based assays where instrumentation can provide rapid data collection, analysis, and interpretation.
A variety of sample sources can be used to find potential leads, and the source and characteristics of the samples to be tested will dictate how they are handled.
Synthetic compounds and extracts can be robotically prepared, usually by a central weighing facility. Typically a workstation measures the weight of a sample dispensed into a test tube and calculates the proper solvent volume to add for a desired concentration. Because good solubilization is important for best results, methods to homogenize insoluble samples should be included in any laboratory workstation performing this function. Robotic systems with integrated compound weighing, dissolution, and microplate distribution are particularly useful for this laborious and repetitive task. Typically, master microtiter racks are created, and daughter plates are prepared based upon the individual needs for each screening group. Using automated pipetting devices or liquid handling systems, a specific amount of sample is distributed into microtiter plates having the well density of choice, and samples can be dried by allowing the solvent to evaporate in a fume hood. The dried sample plates can then be distributed to different screening areas without fear of compound spillage. A number of commercially available robotic systems both large and small are available for compound dissolution and storage.
High-throughput agar-based assays can be readily performed with high efficiency. Large bioassay plates containing agar seeded with an appropriate recombinant test organism such as S. cerevisiae, E. coli, or filamentous fungi can quickly be prepared . Use of these bioassay plates has the advantage of allowing a large number of samples to be tested with one batch of agar, thus minimizing variation in the test organism seed across plates. Both single and multiple plate sets can be prepared according to the particular target of interest. The large capacity assay plates also have the added benefit of allowing the addition of controls outside of the sample array.
Historically, both small and large bioassay plates have been used in industry to create a matrix of sterile paper disks upon which samples were dispensed, typically not exceeding 20 |L per disk, because greater amounts would produce disk saturation leading to sample running and cross-contamination.
Sample application can be carried out more efficiently in a variety of ways. For example, a 96-well cloning device can be routinely used for spotting agar test plates with small amounts of concentrated sample. This step can be performed either manually or robotically depending upon the needs and financial resources of the laboratory. A large number of samples can quickly and accurately be deposited on an agar surface in up to six 96-well arrays for a total of 576 samples per bioassay plate (Fig. 2). Cloning devices can be purchased in 96 pin and 384-well pin configurations for higher density applications [2,304 samples]. Since
Figure 2 High-density agar spotting of 576 samples using a 96-pin cloning device. (Courtesy of J. C. Walsh, American Cyanamid Company, Princeton, NJ.)
concentrated samples are applied, very small sample volumes (1-10 |L) can be spotted without running on the agar surface. By altering the pin design, the dispense volume can be tailored to meet a variety of screening specifications. Samples applied with a cloning device readily absorb into the agar, thus minimizing cross-mixing of samples. After spotting, the assay plates are incubated as required by each assay protocol and scored accordingly. Caution must be exercised when using high-density arrays, since sample toxicity or robust active responses have the potential to mask activity. Retesting of samples within these areas is necessary to identify which sample is responsible for the response.
The basic methodology for conducting microbial-based agar diffusion assays for natural products testing is identical to that for synthetics. Natural products samples, which can potentially contain multiple components in low concentrations, are evaluated using as large a volume of sample as is practical, to maximize the chance of active identification. Wide-bore pipette tips are used for sample distribution to prevent clogging by mycelial fragments and debris that are present in whole broth samples. This is of less concern when testing natural products extracts. In order to maximize the amount of sample applied for testing, agar wells (5 mm) can be bored into an agar surface in an array suitable for high-density testing, and the test wells can be filled manually or using a robotic system . This method allows for significantly larger amounts of sample to be tested than is possible using a standard 1/4 inch paper disk. The filled assay plates are then incubated as required and scored according to the criteria established for each assay. Because of the possibility that motile organisms contained in natural products whole broth samples can spread across the agar test surface obscuring the results, appropriate antibiotics can be added to the agar medium. The antibiotic or combination of antibiotics must control contamination without being toxic to the test organism. Minimum inhibitory concentrations must be determined for each antibiotic against the test organism used .
One of the benefits of using microbial-based assays for novel drug discovery is the flexibility to perform HTS in either agar diffusion assays or liquid assays. Liquid assays are particularly amenable to miniaturization and robotic processing. Advances in miniaturization of labware and the development of new and improved high-density microplate arrays provide an effective means of conducting high-throughput screening. Ultra-high-throughput screening rates of 100,000 compounds per day are now achievable using state-of-the-art microplate robots. Liquid microplate assays incorporating spectrophotometric, fluorescent, or chemiluminescent end points allow rapid quantification and the ability to provide immediate data for analysis and reporting.
The screen development process is the first step in the discovery process. Mechanism-based assays offer many distinct advantages over conventional ''spray and pray'' methods. Mechanism-based screens should be rapid and inexpensive to perform and should identify compounds that act on the target of interest. Screen sensitivity is extremely important, because limitations are frequently imposed on the quantity of material available for testing. In order to achieve maximize productivity, assays should give clear, robust responses with a minimum of assay interference. Assays that are easy to score offer a greater potential for higher throughput even if scoring is performed without the aid of instrumentation.
E. Use of High-Density Agar Diffusion Assays for Assay Validation
Before implementing a new assay, it is helpful to characterize the assay by observing how it responds when tested against various chemical classes. Libraries consisting of thousands of diverse chemical samples can be maintained exclusively for the purpose of validating new assays. High-density agar diffusion or liquid assays can be quickly performed to characterize a new assay prior to implementation. Using agar assays, validation can be quickly accomplished in either 96- or 384-well format. Utilization of a 384-well format can greatly reduce the sample storage requirements for the collection. Automated liquid handling devices can be used to create high-density storage plates from vials or lower density well formats. Likewise, automated pipetting devices can be used for conducting high-density liquid assays.
Of paramount importance during the development phase of any high-throughput microbial assay is the prerequisite that the assay be adaptable to laboratory automation. Next, screen development and screen implementation personnel must work as a team to optimize the assay to achieve maximum throughput. Laboratory robotics can provide many benefits over manually performed techniques because automated devices are capable of carrying out tasks with a high degree of preci-sion-thus achieving quality of results. Incorporation of modular automated devices in the laboratory allows for maximum application flexibility. Using generic workstations and easy programming techniques, screening applications can be quickly modified as needed. The use of laboratory robotics can minimize the exposure of workers to potentially hazardous materials. Application logging and sample tracking using bar code readers provide an accurate means for data auditing. In situations where common screening applications are carried out in a company across divisions, duplication of laboratory robotics systems can be beneficial due to the potential for corporate standardization.
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