Magnetic Bead Based Immunoassay for Autonomous Detection of Toxins

Journal Article · Thu May 01 04:00:00 EDT 2008 · Analytical Chemistry, vol. 80, no. 22, November 15, 2008, pp. 8416-8423

DOI:https://doi.org/10.1021/ac8010044· OSTI ID:946259

Kwon, Y; Hara, C A; Knize, M G; Hwang, M H; Venkatesteswaran, K S; Wheeler, E K; Bell, P M; Renzi, R F; Fruetel, J A; Bailey, C G

As a step towards toward the development of a rapid, reliable analyzer for bioagents in the environment, we are developing an automated system for the simultaneous detection of a group of select agents and toxins. To detect toxins, we modified and automated an antibody-based approach previously developed for manual medical diagnostics that uses fluorescent eTag{trademark} reporter molecules and is suitable for highly multiplexed assays. Detection is based on two antibodies binding simultaneously to a single antigen, one of which is labeled with biotin while the other is conjugated to a fluorescent eTag{trademark} through a cleavable linkage. Aqueous samples are incubated with the mixture of antibodies along with streptavidin-coated magnetic beads coupled to a photo-activatable porphyrin complex. In the presence of antigen, a molecular complex is formed where the cleavable linkage is held in proximity to the photoactivable group. Upon excitation at 680 nm, free radicals are generated, which diffuse and cleave the linkage, releasing the eTags{trademark}. Released eTags{trademark} are analyzed using capillary gel electrophoresis with laser-induced fluorescence detection. Limits of detection for ovalbumin and botulinum toxoid individually were 4 ng/mL (or 80 pg) and 16 ng/mL (or 320 pg), respectively, using the manual assay. In addition, we demonstrated the use of pairs of antibodies from different sources in a single assay to decrease the rate of false positives. Automation of the assay was demonstrated on a flow-through format with higher LODs of 125 ng/mL (or 2.5 ng) each of a mixture of ovalbumin and botulinum toxoid. This versatile assay can be easily modified with the appropriate antibodies to detect a wide range of toxins and other proteins.

• Research Organization: Lawrence Livermore National Laboratory (LLNL), Livermore, CA
• Sponsoring Organization: USDOE
• DOE Contract Number: W-7405-ENG-48
• OSTI ID: 946259
• Report Number(s): LLNL-JRNL-403909
• Journal Information: Analytical Chemistry, vol. 80, no. 22, November 15, 2008, pp. 8416-8423, Journal Name: Analytical Chemistry, vol. 80, no. 22, November 15, 2008, pp. 8416-8423 Journal Issue: 22 Vol. 80
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ANTIBODIES
AUTOMATION
BIOTIN
DETECTION
ELECTROPHORESIS
EXCITATION
FLUORESCENCE
IMMUNOASSAY
MIXTURES
OVALBUMIN
PORPHYRINS
PROTEINS
RADICALS
TOXINS
TOXOIDS