Navigation Links
Counting Non-Aqueous Samples in the TopCount Microplate Scintillation and Luminescence Counter

Abstract

The microplate format has become a standard for many biological assays such as high throughput screening for drug discovery and radioimmunoassays. Productivity with these high volume procedures has been limited by the amount of sample handling required and low sample counting throughput. Now, however, microplate assays can be efficiently counted in microplates with the Packard TopCount Microplate Scintillation and Luminescence Counter. This paper summarizes TopCounts performance for counting samples in organic solvents or bound to the wells of microplates (in-plate assays) using MicroScintTM-O scintillation cocktail, a special formulation for samples in organic solvents. Results from an enzyme inhibition assay and a microplate immunoassay obtained with TopCount are comparable to those obtained by conventional liquid scintillation and gamma counting.

Introduction

Enzyme inhibition assays are among the many tests used by pharmaceutical companies to screen new compounds for biological activity. These assays measure the inhibitory effect of test compounds on target enzymes such as HIV proteases, various kinases, RNA and DNA transcriptases and polymerases. Enzyme activity is often determined by measuring the conversion of a radiolabeled substrate to a product that can be separated from the substrate by chromatography or organic extraction. After the separation step, the 3H- or 14C-labeled products, often in organic solvents, are counted by liquid scintillation counting (LSC). The assays are often done in microplates, and it is highly desirable to quantitate the radiolabeled product in this format, especially when the product can be eluted directly into microplates. By quantitating in microplates, additional sample handling and liquid scintillation vials can be eliminated. In addition, sample and cocktail volumes, and the resulting waste disposal costs, can be reduced significantly. Keeping samples in microplates increases throughput and decreases processing time.

Immunoradiometric assays (IRMA) are often processed in microplates. Typically, this involves a sandwich assay in which the analyte binds to a first antibody which is immobilized in the wells of a microplate or a microplate strip. After washing, the analyte captured in the wells is detected by incubation with a second 125I-labeled antibody. The 125I label in the wells after further washing may be quantitated by transferring individual microplate wells to tubes in a gamma counter. Counting immunoassays directly in the microplates substantially decreases the sample handling.

To facilitate high throughput analysis Packard Instrument Company has introduced the TopCount Microplate Scintillation and Luminescence Counter for counting organic or aqueous samples in either 96-well or 24-well microplates. Specially designed, solvent resistant microplates, PicoPlatesTM, are provided for efficient scintillation counting. Polystyrene microplates are dissolved by many common opaque solvents. Up to 12 wells of a 96-well microplate can be counted simultaneously. TopCount can count an entire microplate in a fraction of the time it takes for conventional liquid scintillation or gamma counting. The available microplate stackers hold 20 or 40 plates for unattended, high throughput processing. The on-line data reduction capability of TopCount can further speed the process of turning raw data into final answers.

In the experiments reported here, TopCount was used to count samples in organic solvents or on a solid support in the microplate format. The scintillation cocktail used was MicroScint-O, an environmentally safe formulation for counting samples in TopCount with most commonly used organic solvents such as heptane, ethyl acetate, methanol, and ethanol. Counting efficiencies were determined using 3H and 14C hexadecane for both the 96- and 24-well formats. To test the effects of sample load, varying volumes of samples in an organic solvent from an enzyme inhibition assay were counted in 96-well solvent resistant PicoPlates, and the results were compared to those obtained by conventional LSC. A TSH immunoradiometric assay was counted, and the results were compared to those obtained by gamma counting.

Experimental Methods

Counting efficiency was tested by adding 3H hexadecane to three wells of a 96-well PicoPlate containing 250 μL of MicroScint-O. For the 24-well PicoPlate, 3H was added to three wells containing 1.0 mL of cocktail. The same number of wells containing only cocktail were used for background determination. Similar experiments were carried out for 14C. The plates were sealed with Packards TopSealTM-P, a solvent resistant cover film, using the Packard MicroMate 496 Microplate Heat Sealer, and then mixed on a microplate shaker for 15 minutes. The samples were counted for five minutes each, under conditions optimized for liquid counting, on a TopCount with the VariPlateTM option which allows counting of either 96- or 24-well plates. To test the effects of sample load, 3H labeled samples in heptane solvent from an enzyme inhibition assay were counted using four different sample volumes. Aliquots of 25, 50, 100, and 150 μL in quadruplicate were pipetted into a solvent resistant 96-well Pico- Plate with a multiple-tip pipetter. Total volumes were brought to 300 μL with MicroScint-O. The plates were sealed and counted as described above. An additional 50 μL aliquot was counted for five minutes in a Packard Tri-Carb 2250CA liquid scintillation analyzer, using 5 mL Opti-Fluor organic cocktail in 7 mL glass vials, and the results were correlated with those from TopCount. Dose curves for the enzyme inhibitor and a control sample value obtained with TopCount were compared with those from the Packard Tri-Carb 2250CA.

A sandwich immunoradiometric assay (IRMA) for TSH was performed in a solvent resistant PicoPlate or in microplate strips (Dynex Technologies) coated with anti-TSH MAb as the first antibody. An 125Ilabeled anti-TSH MAb was used to quantitate TSH bound to the solid support by the first antibody. The PicoPlate was counted in the TopCount with 250 μL MicroScint-O, and individual wells of the microplate strip were counted in a Packard Cobra gamma counter for comparison.

Results and Discussion

The average efficiencies and backgrounds obtained with TopCount with a small sample load are presented in Table 1. A small sample (10 μL) was used to provide the optimum cocktail performance. Counting efficiencies for 3H are comparable to those which can be obtained by conventional liquid scintillation counting.

The CPM results for equal volumes of 3H labeled samples in heptane counted with TopCount and a conventional LSC are compared in Figure 1. The data show that the TopCount correlates very well with the LSC (R2= 0.99). The slope of the line indicates that for this sample load TopCount CPM is approximately 40% of the CPM obtained by conventional LSC. This lower efficiency is a result of the much higher sample load; only 250 μL of cocktail was used in TopCount versus 5 mL in the LSC.

Table 2 shows a comparison of the % inhibition values for a control sample obtained from TopCount with the value obtained by LSC. Assays 1-4 were counted with TopCount using four different sample volumes, and assay five was counted with the Tri- Carb 2250CA. These control values are additional confirmation that TopCount provides consistent results regardless of the sample load used in the assay. Table 2. Comparison of inhibition values obtained from TopCount and LSC.

The results of an 125I-labeled IRMA for TSH counted by liquid scintillation counting on TopCount and on a gamma counter are shown in Figure 3. The standard curves are essentially the same for the two methods of quantitation. Direct counting of immunoassay microplates with TopCount eliminates the need to transfer individual microplate wells to tubes for counting in a gamma counter. The counting efficiency for 125I in TopCount, using MicroScint-O, is higher than 50%.

Conclusions

The Packard TopCount Microplate Scintillation and Luminescence Counter, PicoPlates, and the specially formulated scintillation cocktail, MicroScint- O, provide results from samples in organic solvents or on solid supports equivalent to those obtained with a conventional liquid scintillation counter or gamma counter. Up to 50% sample loads of organic solvents may be used. With small sample loads, counting efficiencies are comparable to conventional LSC. For assays prepared in the microplate, TopCount provides quantitation in the same format. This results in increased throughput, less sample handling, and significant savings in reagent and waste disposal costs.

Acknowledgements

The enzyme inhibition samples were supplied by Dr. Russell Lingham; Merck, Sharpe, and Dohme; Rahway, New Jersey, U.S.A. The TSH immunoassay work was performed by Dr. Alain Baret; Labratoire D'Analysis Medicales; Nantes, France.


'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Counting Colonies or Plaques Directly from Images of Agar Plates
2. Counting Cytostar-T scintillating microplates on the MicroBeta JET
3. Counting FlashPlates on the Wallac MicroBeta
4. Cerenkov Counting Performance on the TopCountTM Microplate Scintillation and Luminescence Counter
5. Direct Counting of Millipore MultiScreen Filtration Plates.
6. New Automated Yeast Cell Counter Ends Tedium & Errors of Manual Counting
7. Quantitation of the Protein MIA as a Marker for Chondrocytes in Research Samples
8. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
9. Preserve Samples for RNA Expression Microarrays RNAlater Around the World
10. RNA from LCM Samples
11. Robust High Throughput RNA Isolation from Blood Samples
Post Your Comments:
(Date:12/25/2014)... (PRWEB) December 25, 2014 The report ... by Product Type & Provider Type - Global Advancements, ... market with an in-depth analysis and forecasting of revenues. ... spread through 221 pages and in-depth TOC on “Service ...
(Date:12/24/2014)... , Dec. 24, 2014  Vermillion, Inc. (Nasdaq:   ... disease, announced today that the Company closed its ... Investment Management, Jack W. Schuler , Birchview ... were $10.5 million, before offering expenses.  The proceeds ...
(Date:12/24/2014)... 2014 GMO corn cases filed across the ... the process of being consolidated in a Kansas federal court ... AG MIR 162 Corn Litigation, MDL No. 2591 in the ... of the Syngenta GMO corn multidistrict litigation (MDL) has been ...
(Date:12/24/2014)... MD (PRWEB) December 23, 2014 On ... H.R. 83, the Omnibus and Continuing Resolution Appropriations Act ... a condition eligible to receive funding through the Congressionally ... of Defense (DoD). The Hydrocephalus Association (HA), working in ...
Breaking Biology Technology:Service Quality Management Market worth $2.1 Billion & Telco Customer Experience Management Market worth $2.3 Billion by 2019 – New Report by MarketsandMarkets 2Service Quality Management Market worth $2.1 Billion & Telco Customer Experience Management Market worth $2.3 Billion by 2019 – New Report by MarketsandMarkets 3Service Quality Management Market worth $2.1 Billion & Telco Customer Experience Management Market worth $2.3 Billion by 2019 – New Report by MarketsandMarkets 4Vermillion Announces Closing of Equity Financing 2Vermillion Announces Closing of Equity Financing 3Carey Danis & Lowe Reports on Syngenta GMO Corn Transfer Order 2Rare Brain Condition Now Eligible for Department of Defense Research Dollars 2
... ,, CAMBRIDGE, England , May 25, ... ( Cambridge, UK ) announced today,an extension of the agreement under which ... , , ... Miles Congreve , Head of Chemistry said, "We are happy to ...
... China , May 25 /PRNewswire-Asia-FirstCall/ -- Biostar,Pharmaceuticals, Inc. (Nasdaq: ... leading over-the-counter Hepatitis B medicine,Xin Aoxing Oleanolic Acid Capsule ("Xin Aoxing"), ... it has,commenced assembly of its production line for its proprietary Zushima ... ...
... May 25 Fibrocell Science, Inc. (OTC Bulletin ... autologous cell therapies for aesthetic, medical and scientific applications, announced ... Irish Voice newspaper and Irish America Magazine ... Officer Declan Daly as a member of the ...
Cached Biology Technology:Heptares Therapeutics Extends Multi-FTE Chemistry Collaboration With Oxygen Healthcare (O2h) 2Heptares Therapeutics Extends Multi-FTE Chemistry Collaboration With Oxygen Healthcare (O2h) 3Biostar Pharmaceuticals, Inc. Begins Assembly of New Production Line 2Biostar Pharmaceuticals, Inc. Begins Assembly of New Production Line 3Biostar Pharmaceuticals, Inc. Begins Assembly of New Production Line 4The Irish Government and the Irish Voice Recognize Fibrocell Science, Inc.'s Declan Daly as 'Irish Life Science 50' Member 2
(Date:1/22/2015)... 2015 , Europe,s ... The European Patent Office to present a video retrospective of the ... be featured: Christofer Toumazou and Sophie Wilson   ... winners of the Award   Starting on 22 January ...
(Date:12/22/2014)... 2014  NXT-ID, Inc. (NASDAQ: NXTD and NXTDW) ... the growing mobile commerce market, announces it has retained famous ... the 2015 International CES debut of the Wocket™ biometric smart ... at the NXT-ID booth January 6th and 7 th , ...
(Date:12/19/2014)...  23andMe, Inc., the leading personal genetics company, today announced the ... ancestry of individuals from across the United States ... four hundred years ago, the United States ... different continents. This study illuminates how American history and the ...
Breaking Biology News(10 mins):Ten Years of the European Inventor Award: A Retrospective Look at the Inventors and Ideas That Have Changed Our Lives 2Ten Years of the European Inventor Award: A Retrospective Look at the Inventors and Ideas That Have Changed Our Lives 3NXT-ID Hires Master Pickpocket and Security Consultant Apollo Robbins for Wocket Smart Wallet CES 2015 Debut 2NXT-ID Hires Master Pickpocket and Security Consultant Apollo Robbins for Wocket Smart Wallet CES 2015 Debut 3NXT-ID Hires Master Pickpocket and Security Consultant Apollo Robbins for Wocket Smart Wallet CES 2015 Debut 423andMe Study Sketches Genetic Portrait of the United States 223andMe Study Sketches Genetic Portrait of the United States 323andMe Study Sketches Genetic Portrait of the United States 4
... led by Chu Chen, PhD, Associate Professor of Neuroscience at ... called Monoacylglycerol lipase (MAGL) as a new therapeutic target to ... November 1, 2012 in the Online Now section of the ... inactivation of MAGL, best known for its role in degrading ...
... delves into the unexpected properties of life at the nanoscale ... Molecular Machines Extract Order from Chaos," by Peter M. Hoffmann, ... in Wayne State University,s College of Liberal Arts and Sciences, ... and pulleys, atoms and quarks to explain life. ...
... has found that the sensitivity and recovery of UK butterfly populations ... of fragmentation of key habitat types in the landscape. ... , used data on the Ringlet butterfly collected from 79 UK ... spanned a severe drought event in 1995. The study ...
Cached Biology News:LSUHSC research identifies new therapeutic target for Alzheimer's disease 2New book on physics principles by Wayne State professor explains life as we know it 2UK butterfly populations threatened by extreme drought and landscape fragmentation 2
... Ocean Optics LIBS2000+ Spectrometers (sold separately) in ... Ablation Chamber to offer numerous configuration options ... features include a high-power 1064 nm Nd:YAG ... X-Y sample positioning, laser spot diameter control ...
... Kodak Molecular Imaging Systems offer an ... life science researchers to conveniently capture ... tissues, organs and whole animals with ... localization of molecular signals that are ...
... Cleanroom-grade vinyl curtains or ... powder-coated steel support frame to ... cleanroom that meets cleanliness to ... HEPA filter/fan units, lights, and ...
... first high-density serum biomarker chip to profile ... An antibody chip containing 120 human cancer ... enables biomarker discovery scientists to study panels ... of therapy and the likelihood of recurrence ...
Biology Products: