Emerging Labeling Technologies
المؤلف:
Mary Louise Turgeon
المصدر:
Immunology & Serology in Laboratory Medicine
الجزء والصفحة:
5th E, P165-167
2025-07-22
690
Quantum Dots (Q dots)
An advanced labeling technique, quantum dots are semiconductor nanocrystals used as fluorescent labeling reagents for biological imaging. A valuable property of Q dots is that different sizes of crystals produce different signals with a single laser excitation. This seemingly simple physical property implies that different-sized Q dots could be directed against different analyte targets, and the Q dots would fluoresce with different colors in a size-dependent manner. This allows for the detection of multiple analytes with a single assay. Q dots are the next step in the evolution of luminescence-based assays.
SQUID Technology
A novel method of target labeling is to tag antibodies with superparamagnetic particles, allow the tagged antibodies to bind with the target antigen, and use a superconducting quantum interference device (SQUID) to detect the tagged antigen antibody complex. The amplitude of the signal is proportional to the number of bound particles and correspondingly to the amount of target. A current application of this technology is its use in the detection of Listeria monocytogenes.
Luminescent Oxygen-Channeling Immunoassay
This novel detection technology is based on two different 200-nm latex particles, a sensitizer particle that absorbs energy at 680 nm with the generation of singlet oxygen (donor bead) and a chemiluminescer molecule that shifts the emission wavelength to 570 nm (receptor bead). When these particles are in proximity during excitation, singlet oxygen moves from the donor bead to the receptor bead, where it triggers the generation of a luminescent signal. Luminescent oxygen-channeling immunoassay (LOCI) technology is broadly applicable to any molecule that can be determined in a binding assay. The production of end point ribonucleic acid (RNA) determination by LOCI has been investigated.
Signal Amplification Technology
Tyramide signal amplification (TSA) can be used in various f luorescent and colorimetric detection applications. TSA pro tocols are simple and require few changes to standard operating procedures. TSA provides a messenger RNA (mRNA) in situ hybridization protocol that is effective in detecting B cell clonality in plastic-embedded tissue specimens. Immunoglobulin light-chain mRNA molecules can be detected directly in paraffin-embedded tissue using fluorescein-labeled oligonucleotide probes. TSA amplification enables B cells to be detected in tissue sections without additional processing steps and specially prepared sections. Similar in situ hybridization technology can also be used for the detection of cytokines, such as interferon gamma (IFN-γ) and interleukin-4 (IL-4).
Magnetic Labeling Technology
Magnetic labeling technology is an application of the high- resolution magnetic recording technology developed for the computer disk drive industry. Increased density of microscopic, magnetically labeled biological samples (e.g., nucleic acid on a biochip) translates directly into reduced sample-processing times. Magnetic labeling can be applied to automated DNA sequences, DNA probe technology, and gel electrophoresis (Fig. 1). Compared with other nonradioactive labeling systems, magnetic labels are inherently safe, instrumentation is less expensive, signals are almost permanent, and spatial resolution is increased.
Fig1. Magnetic labeling techniques. A, Detection of deoxyribonucleic acid. B, Detection of antibodies. (Adapted from Adelman L: Labora tory technology: magnetic labeling technology, Adv Med Lab Admin 11:131, 1999.)
In a magnetic label–based gel electrophoresis application sphere, DNA is analyzed. DNA is separated into bands using electrophoresis and magnetic labels are bound to the DNA in each band. By applying and then removing a magnetic field, the magnetic domains in each label are oriented in the same direction, resulting in a net magnetic field near the bands in the direction of the applied field (Fig. 2).
Fig2. Cross-sectional schematic of small region of sequencing gel or nylon membrane with magnetic labels bound to DNA, separated into two bands. Left, Arrows on band represent the magnetic field resulting from the magnetized labels. Right, Band has a sensor near the surface. (Adapted from Adelman L: Laboratory technology: magnetic labeling technology, Adv Med Lab Admin 11:131, 1999.)
Time-Resolved Fluoroimmunoassay
In a time-resolved assay, fluorescence is measured after a certain period to exclude background interference fluorescence. This form of immunoassay is heterogeneous with a direct format (sandwich assay), similar to direct ELISA. The time resolved fluoroimmunoassay uses europium-labeled anti bodies. If excited at 340 nm, europium fluoresces at 620 nm. The fluorescence is measured and is directly proportional to the concentration of the substance.
Fluorescence Polarization Immunoassay
In the fluorescence polarization immunoassay, a homogeneous competitive fluoroimmunoassay, the polarization of the fluorescence from a fluorescein-antigen conjugate is determined by its rate of rotation during the lifetime of the excited state in solution. Binding to a large antibody mole cule slows down the rate of rotation and increases the degree of polarization, and the fluorescence emitted is polarized.
Fluorescence in Situ Hybridization
Fluorescence in situ hybridization (FISH) uses fluorescent molecules to brightly “paint” genes or chromosomes. The rapid expansion in the availability of polyclonal and mono clonal antibodies has fostered a dramatic increase in light microscopic immunohistochemistry (IHC) and in situ hybridization.
The FISH molecular cytogenetic technique uses recombinant DNA technology. Probes are short sequences of single stranded DNA that are complementary to the DNA sequences to be examined. Probes hybridize, or bind, to the complementary DNA (cDNA) and labeled fluorescent tags indicate the location of the sequences. Probes can be locus specific, centromeric repeat probes, or whole-chromosome probes.
In metaphase FISH, a specific nucleic acid sequence (probe) is bound to the homologous segment on a metaphase chromo some affixed to a glass slide. Uniquely, the existence of a region specific DNA sequence in a nondividing cell can be detected using interphase FISH.
Clinical applications of FISH for the detection of inherited and acquired chromosomal abnormalities include hematopathology and oncology. Many genetic syndromes have been recognized by geneticists, but laboratory tests often are unavailable for confirmation. The DiGeorge syndrome is an example of a chromosomal deletion leading to the loss of several genes.
A simple sensitive method for in situ amplified chemiluminescent detection of sequence-specific DNA and IgG immunoassay has been developed. This immunoassay uses highly active gold nanoparticles as the label and can be confirmed by clinical testing. The method has many desirable features, including rapid detection, selectivity, and minimal instrumentation. The protocol has potentially broad applications for clinical immunoassays and DNA hybridization analysis.
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