Nanophosphor Lateral Flow Assay for Detecting HSV-2

INTRODUCTION:
Human Herpes Simplex Type 2 (HSV-2) causes a life-long infection and occurs in over 10% of adult individuals. This report details the approach by Goux et al. (2019) to identify HSV-2  infections using a Smartphone to detect the nanophosphor signal of a lateral flow device.

DISCUSSION:
Lateral flow assays (LFAs) are needed to improve the detection of HSV-2 without the time, cost, and lack of privacy associated with a laboratory setting. As of December 2019, there are no commercially available gold nanoparticle LFAs for HSV-2. Laderman et al. (2008) described the development of a gold nanoparticle-based immunoblot test for HSV-2. This assay, called Sure-Vue HSV-2 Rapid test, had a sensitivity of 94% and a specificity of 98%. Goux et al. (2019) developed the assay to improve the clinical sensitivity and specificity.

To do this, Paterson et al. (2004) noted they used strontium aluminate persistent luminescent nanoparticles, which they had previously developed (PLNPs, nanophosphors) as the LFA reporters. These PLNPs have a long-lasting, bright glow excitation, which allows for a delay of emission measurement, reduced background autofluorescence, and eliminates the need for precision optical filters. The strontium aluminate doped with europium and dysprosium has a bright, long-lasting light emission, which is inexpensive and widely used in “glow-in-the-dark” signs and toys. Paterson et al. (2014) stated that strontium aluminate PLNP LFAs showed to have a higher analytical sensitivity than traditional LFAs. Goux et al. (2019) tested a panel of 21 human plasma and serum samples ranging from negative to strongly positive for HSV-1 and HSV-2 (PTH2020; SeraCare Life Science). Then, they mixed 20μl of human plasma or serum samples (10μl of sample + 25μl of buffer) with 15μl of anti-human IgG-PLNP conjugate. Finally, they dispensed the sample/nanophosphor mixture (35 μl) onto the LFA pad. Goat anti-human IgG nanophosphors bearing anti-HSV human IgG (if present in the sample) migrated up the membrane. A recombinant HSV-2 antigen immobilized at the test line captured the
nanophosphors. Unbound anti-human nanophosphors bearing human IgG migrated further up the strip until captured by the goat anti-human IgGs immobilized at the control line.

A custom LFA iPhone app on an iPhone 7 Plus Smartphone (Apple Inc.) in combination with a 3D-printed attachment imaged the LFA strips. The PLNPs were excited by turning on the iPhone light (4s, maximum intensity) and then cycling the camera’s flash. After a delay (~100ms after excitation), the phone camera acquired an image of phosphor emission. Researchers repeated the excitation/imaging cycle four times, and the four images were stacked together to reduce background noise and increase reproducibility. They were able to detect between 5.7 to 23.3 mg/ml of human IgG. They detected no crossreactivity to HSV-1 in 10 tested samples. For comparison, the LFA test strips were also imaged on a FluorChembased imaging platform with two 10W ultraviolet LED lights (395-400nm) and a CoolSNAP K4 CCD 2,048 x 2,048-pixel camera. PLNP were excited with the LEDs for 1 min and imaged with an exposure time of 1s and pixel binning of 4.

CONCLUSION:
The nanophosphor HSV-2 LFA had a sensitivity of 96.7%, with 100% specificity for detecting HSV-2 in the tested samples. This sensitivity was higher than that of commercially available rapid HSV-2 assays tested with the same panel. This smartphone-based nanophosphor LFA technology shows promise for private self-testing for sexually-transmitted infections.

By David Kilpatrick, PhD and Abbas Vafai, PhD