NEW YORK (360Dx) – Researchers at China's National Institute of Biological Sciences (NIBS) have developed an immunofluorescence amplification technique that could significantly boost the sensitivity of some immmunoassays.
Described in a paper published this week in Nature Methods, the approach combines conventional antibody-based detection with hybridization chain reaction to amplify the fluorescent signal produced upon antibody-target binding by as much as two to three orders of magnitude.
Called immunosignal hybridization chain reaction (isHCR), the method is broadly applicable to a variety of immunoassay formats and could prove useful in areas ranging from clinical pathology to immunoassay-based research, said Minmin Luo, a NIBS investigator and senior author on the study.
Immunoassays are the most commonly used method for protein detection and quantification. In most immunoassays, an antibody binding to its target produces a visual signal such as fluorescence which can be amplified via enzyme-based reactions and then read by researchers.
Enzyme-based signal amplification is "very useful and widely-employed," Luo said, but the approach has a number of limitations.
For instance, he said, "they often generate high background and can reduce spatial resolution due to dye diffusion." Additionally, "they lack the capacity for high multiplexity, which currently excludes their application in high-throughput, systematic studies," he added.
They are also not compatible with newer tissue expansion techniques in which researchers use polymers to physically expand a sample of interest, allowing them to investigate it at higher resolution.
To address these limitations, Luo and his colleagues looked to hybridization chain reaction technology, which has been used to amplify signal in nucleic acid assays like fluorescence in situ hybridization (FISH). HCR uses pieces of hairpin nucleic acids mixed with a single-strand nucleic acid initiator to launch a chain reaction that will continue growing so long as nucleic acid hairpins are present. Signal amplification is achieved by labeling these hairpins with fluorophores.
The NIBS researchers adapted this approach to antibody-based assays by biotinylating the detection antibodies and the HCR initiator. Upon the addition of streptavidin, the antibody-antigen complex is linked to the HCR initiator. Researchers can then add fluorophore-labeled hairpins to amplify the detection signal.
The isHCR could allow for more sensitive detection of protein targets, Luo said, noting that while antibody specificity and binding affinity are "the critical factors for the performance of immunoassays," approaches like immunohistochemistry can fail to detect low abundance targets even with sufficiently sensitive antibodies due to a lack of the necessary signal amplification.
"Monoclonal antibodies are an example of this situation," he said. "Despite their superior properties compared with traditional polyclonal antibodies, such as higher specificity, and being cheaper and easier to produce, they are not used as much because of the low signal they frequently generate."
The approach might also allow researchers to make better use of antibodies with low binding affinity, Luo said. "They can be used at much lower concentration in immunoassays [due to] the signal amplification from isHCR, [which] will likely decrease background noise, and therefore increase the sensitivity of detection."
Luo and his colleagues tested the method in a variety of settings, finding that it improved detection in assays including Western blots and immunohistochemistry of cell and tissue samples. In work with HeLa cells infected with Salmonella enterica serovar Typhimurium (S. Typhimurium) they found the method could detect the S. Typhimurium effectors SteA and SopD2 while standard IHC could not.
The researchers also used the method in samples expanded via expansion microscopy, a process that allows for high resolution studies of cells and tissue samples but causes significant dilution of the fluorescent probes typically used for immunoassay detection. Use of the isHCR boosted signal intensity by three orders of magnitude compared to standard IHC, they noted.
"We tested isHCR in different types of samples including purified proteins, cultured cells, tissue sections, and whole organs," Luo said. "We also tested [its effectiveness] for the detection of different proteins at various cellular positions, and in all cases, we didn’t find any biases in its performance."
The technique could also prove attractive from a multiplexing perspective, he said, noting that in the Nature Methods work he and his colleagues "successfully performed simultaneous detection of three different proteins in a single round of isHCR amplification [using] three different fluorescent molecules that have distinct emission wavelength for the signal detection."
The technique's multiplexing capabilities could be further expanded by incorporating additional fluorescent labels, he said. He added that because the approach uses DNA for signal amplification, the labels can be removed using a deoxyribonuclease to degrade these molecules.
That means "it’s possible to perform [multiple] rounds of isHCR amplification-detection-removal, which will dramatically expand the multiplexity of isHCR," Luo said.
The researchers also explored the use of multiple rounds of amplification to provide a further increase in signal. In this case, the hairpin DNA amplifiers are also biotin-tagged. This allows addition of another streptavidin molecule, which can then bind another biotin-tagged isHCR initiator, launching another round of signal amplification.
In the Nature Methods work, Luo and his colleagues performed three rounds of amplification, finding that in Western blotting experiments the two additional rounds boosted the assay's sensitivity tenfold.
Luo said the technique could in theory go beyond three rounds of amplification, though he noted that the researchers have not determined what the practical limit of this approach is.
While the technique is broadly applicable, Luo suggested that "a perfect niche for isHCR would be IHC-based pathology," where it could allow clinicians to multiplex detection of protein markers in the same pathological specimen as well as measure low abundance markers not accessibly with standard IHC.
He added that for applications like ELISA, where sensitivity of detection is more important than spatial resolution, the technique could be combined with conventional enzymatic reactions to achieve even higher signal amplification.
Luo said he and his colleagues have filed patents around applications of the technology and are interested in commercializing the method, though he did not provide any specifics on their plans.