to BioTechniques free email alert service to receive content updates.

protocol
Automated High-throughput Insulin Detection Using AlphaLISA Assay and JANUS Automated Workstation
Sponsored,vendor-submitted protocol   Published in November 2009 (p.57) DOI: 10.2144/000113036

PerkinElmer's AlphaLISA® chemistry can be used to assay a number of analytes in such areas as drug discovery, immuno-diagnostics, HT screening, and basic research. AlphaLISA allows for the ability to run large numbers of samples with small sample volume, excellent sensitivity, and an expanded dynamic range relative to ELISA, all at room temperature and without the need for plate washing or shaking. Plate densities of 96, 384, and 1536 wells are accommodated. These characteristics make this chemistry ideal for high-throughput screening and automation with the JANUS® Automated Workstations and detection with the EnVision Multilabel Plate reader. Here, automation of an AlphaLISA insulin detection assay is described, and favorable comparison between automated and manual liquid handling is demonstrated.

Materials

JANUS Mini MDT Automated Workstation (Figure 1) equipped with a P30/384-MDT dispense head and 30 µL P30 clear non-sterile disposable tips.

2103 EnVision Multilabel Plate Reader with Alpha capability

AlphaLISA® Insulin Kit (PerkinElmer) AlphaLISA Acceptor beads coated with an Anti-Analyte Antibody

Streptavidin-coated Donor beads

Biotinylated Anti-Analyte Antibody

Lyophilized analyte

10× AlphaLISA ImmunoAssay Buffer

NOTE: FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Insulin (Fitzgerald Industries)

White OptiPlate™-384 (PerkinElmer)

Protocol

The general AlphaLISA assay protocol is segmented into 4 steps. All liquid handling steps (including serial dilutions) were carried out on the JANUS Automated Workstation. A general outline of each protocol step is provided in the full protocol.

Prepare AlphaLISA Assay

  1. Preparation of 1× AlphaLISA Immuno-Assay Buffer

  2. Preparation of human insulin analyte standard dilutions

  3. Preparation of 2.5× AlphaLISA Anti-Insulin Acceptor Beads + Biotinylated Antibody Anti-Insulin MIX

  4. Preparation of 2× Streptavidin (SA) Donor beads (80 µg/mL)

Prepare Insulin Samples in 96- or 384-well Microplates

  1. Add 5 µL of each analyte standard dilution

  2. Add 20 µL of a 2.5× MIX:

  3. AlphaLISA Anti-Analyte Acceptor Beads (10 µg/mL final) and Biotinylated Antibody Anti-Analyte (1 nM final)

  4. Incubate 60 min at 23°C

  5. Add 25 µL 2× SA-Donor beads (40 µg/mL final)

  6. Incubate 30 min at 23°C in the dark

  7. Read using EnVision-Alpha Reader

A standard curve ranging from 0.5 to 10,000 µU insulin/mL was prepared in triplicate in a 384-well OptiPlate on the JANUS, as well as manually. Simulated insulin samples of varying concentrations, within the range of the standard curve, were also prepared and run in parallel to demonstrate assay recovery.

Results and Discussion

For complete assay detail and full data sets, please download the complete protocol. We compared manual to automation performance for the generation of assay standard curves (Figure 2) and the results show that assay performance is equivalent between manual and automated methods. To demonstrate the precision of JANUS automation compared to manual sample pipetting, a coefficient of variation (CV) analysis was performed. Across the analyte concentration range, the precision of JANUS was comparable to manual pipetting, with CVs ranging from 0.39% to 4.40%. The standard curve covered 4.5 orders of magnitude, and the lowest detectable doses, based on a buffer sample plus three standard deviations, were 11.4 and 6.9 µU/mL for the automated and manual runs, respectively.





Correspondence

Address correspondence to PerkinElmer, Inc., 940 Winter Street, Waltham, MA 02451, USA, www.perkinelmer.com/labautomation2; Tel.: 800-762-4000 (US & Canada) or +1 203-925-4602; email: [email protected]

More Information
 
Do you have question about this protocol? Would you like more information about this protocol?
Submit your questions here and Biotechniques will forward your question and contact information from our database to an expert at
Yes, please forward my question, along with my name, title, company, and e-mail address to
BioTechniques will forward your question to the company, who will contact you directly by email.
Request more information
 
Name Question
Job Title
Company
Email
Protocol Categories
rightcontent.jsp called otherwise condition rightcontent.jsp called 222222222222222222222 HOME-RSS
Search Protocols
Search our protocols for your specific technique.
Submit Your Protocols
BioTechniques is always looking for author-submitted protocols related to peer-reviewed papers, so submit your protocols today to our editorial team. Suppliers or advertisers interested in publishing their protocols should also submit.
Click here for more information