Attomole Quantitative Chemiluminescence for Molecular Diagnostics

The study and use of bioluminescence and chemiluminescence have increased dramatically in recent years. Applications range from adenosine triphosphate (ATP)–luciferase assay and cell viability tests to immunoassay, DNA, genomic, and proteomic analyses. Unlike fluorescence systems, there is no need for an exogenous light source, since luminescence is generated from chemical reactions.

Utilizing photon-counting detectors, which count the number of photons generated from a chemical reaction, the luminescence assay has become one of the most sensitive and quantitative optical detection methods. During the past 15 years, nucleic acid (NA) hybridization techniques have become increasingly important as discovery tools and for the clinical diagnosis of genetic disorders and infectious diseases.1–3 These methodologies have benefited greatly from the development of NA amplification techniques that have significantly improved the sensitivity of detection of nucleic acids such as DNA and RNA and their chimeric derivatives. Nucleic acid amplification is suitable for low-abundant gene transcripts and genotyping of rare allele mutations, as well as for the measurement of low-copy-number RNA and DNA pathogens (i.e., bacteria and viruses) in diverse biological sources, particularly in applications that depend on extremely limited nucleic acid samples, such as cancer diagnostics, pharmacogenomics, toxicogenomics, forensics, the food industry, and environmental detection.

Molecular diagnostics and target identification

Figure 1 - Magnetic bead-based DNA hybridization assay with a label HRP for chemiluminescence detection. Once the substrate is added, the reaction produces luminescence.

Figure 2 - The magnetic bead-based sandwich hybridization assay has a detection limit between 1 and 5 attomole of target oligonucleotide and a dynamic range of 3–4 orders of magnitude.

Figure 3 - The LuminMax-C offers attomole sensitivity by quantitative photon counting.

The following section describes a sandwich hybridization assay for quantitative oligonucleotide target detection. The assay combines 1) a magnetic bead-based capture probe, 2) target DNA hybridization, and 3) chemiluminescent target detection. For the assay, 5′ end biotinylated single-stranded capture oligonucleotides (MWG Biotech, Highpoint, NC), representing a 30-bp sequence within the coding region of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was attached to streptavidin-coated paramagnetic beads (Dynal MyOne, Invitrogen Corp., Carlsbad, CA) via the biotin–streptavidin bonding. Magnetic beads (20 μg) were incubated with 20 pg of capture oligonucleotide (5′ biotin-GCT CTT TGC ACT GGT AGA CAG AGA TCT CAT- 3′) in 10 mM Tris–HCl (pH 7.5)/1 mM ethylenediaminetetraacetic acid (EDTA)/2 M NaCl for 20 min at room temperature. Unbound material was washed off, and the beads were resuspended in 20 μL of the same buffer (this can be stored at 4 °C for several months). The assembled capture oligo-beads were washed and resuspended in phosphate-buffered saline (PBS) prior to hybridization with a 30-mer target oligonucleotide with the exact complementary sequence plus carrying a digoxigenin molecule at its 5′ end (5′digoxigenin-ATG AGA TCT CTG TCT ACC ABT GCA AAG AGC -3′) (Figure 1). Hybridization was performed in hybridization buffer (MWG Biotech) for 90 min at 42 °C using a thermal cycler. The hybridized product was subsequently blocked with 2% bovine serum albumin (BSA)/0.1% Tween 20/PBS for 30 min at room temperature before adding 3 mU of a sheep antidigoxigenin polyclonal antibody conjugated to horseradish peroxidase (HRP) (Roche Applied Sciences, Indianapolis, IN) to a final concentration of 0.15 mU/μL. The immunodetected beads were washed four times with 1 mL of 1% Tween 20/PBS and finally with PBS before pelleting and resuspending in 20 μL of PBS for transfer to a black clear-bottom microplate (cat. no. 265301, Nalge Nunc International Corp., Rochester, NY). Chemiluminescent signal enhancement was achieved by adding HRP LuminMax Supersubstrate (Maxwell Sensors, Santa Fe Springs, CA) and recorded (Figure 2) with a LuminMax-Creader (Figure 3).

Figure 4 - The dynamic range of the polyclonal antibody reaches over seven orders of magnitude from 30 mU to 10–5 mU using the same amount of LuminMax Super HRP substrate.

The LuminMax-C reader offers microplate setting for cell biology, molecular biology, and immunology applications in the fields of cancer research, drug development, proteomics, and genomics. It is also suitable for testing antibody activity prior to the use of antibodies in ELISAs such as those utilized modularly for the hybridization sandwich assay described above. The authors tested the sensitivity of the antidigoxigenin antibody in a titration assay using different antibody concentrations, while keeping the amount of chemiluminescent substrate constant (Figure 4). The compact, economical system offers high sensitivity, accuracy, and ease of use. It measures the luminescence intensity in a 96-well microplate (black or white). Because the microplate has a clear bottom, luminescence can be accurately detected from the bottom of the well. Since it uses a state-of-the-art photon-counting multiplier tube as the detector, the system is able to count a single photon generated from the reaction, and thus it can detect very small amounts of analyte in the samples. A CD, with user-friendly software, is provided for easy installation. The system utilizes a PC or notebook as its microprocessor. It is interfaced to a computer by a simple plug-in (USB or serial port) connection. After clicking the “Go” button, the system automatically and quickly scans all of the selected microplate wells and shows the results. The resulting data are displayed as a spreadsheet in Microsoft® Excel (Microsoft, Redmond, WA) format.

References

  1. Qureshi, M.N.; Bolick, D.; Ringer, P.J.; Spagler, F.L.; Zimmerman, G. HPV testing in liquid cytology specimens: comparison of analytic sensitivity and specificity for in situ hybridization and chemiluminescent nucleic acid testing. Acta Cytol. 2005, 49(2), 120–6.
  2. Leal, E.; Jaloma-Cruz, A.R.; Barros-Nunez, P. High sensitivity of chemiluminescent methodology for detection of clonal CDR3 sequences in patients with acute lymphoblastic leukemia. Hematol. Oncol. 2004, 22(2), 55–61.
  3. Patel, R.; Pollner, R.; de Keczer, S.; Pease, J.; Pirio, M.; DeChene, N.; Dafforn, A.; Rose, S. Quantification of DNA using the luminescent oxygen channeling assay. Clin. Chem. 2000, 46(9), 1471–7.

Mr. Ho is a Research Intern, and Dr. Tajbakhsh is a Senior Research Scientist, Maxwell Sensors Inc., 10020 Pioneer Blvd., Ste. 103, Santa Fe Springs, CA 90670, U.S.A.; tel.: 562-801-2088; fax: 562-801-2089; e-mail: info@maxwellsensors.com.

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