FROM LABEL-FREE TO LABELED DETECTION FOR ENHANCING SENSITIVITY
During bioprocessing, detection of host-cell protein, Protein A amounts leached from purification columns, and absence of
live mycoplasma are critical for batch validation. These tests currently are performed with methods orthogonal to label-free
techniques, and often rely on ELISA assays performed by a third-party laboratory. While results are being determined, production
is in a stand-by mode.
Although label-free LSPR does not require sample pretreatment, its sensitivity is restricted to the 10–200 ng/mL range of
detection dependent on the protein being analyzed. To bridge the gap between label-free and labeled technology, LSPR detection
can be enhanced with an enzyme-labeled protein capable of selectively binding to the antigen of interest, such as the low
level contaminants mentioned above. With a single reading platform, it becomes possible to expand levels of detection into
the pico- to femtogram range for an assay time <30 min. The principle of LSPR-labeled detection is illustrated in Figure 3A.
An enzyme, such as alkaline phosphatase, is used to convert a substrate into an insoluble form. After deposition on the LSPR
surface, the plasmon peak position red-shifts by between 20 and 100 nm (Figure 3B).
Figure 3. A) Illustration of the label-free and labeled assay implementation using an LSPR surface. In the labeled case, an
enzyme is linked to a specific protein. Similar to an immunoassay, the enzyme is used to convert a soluble substrate into
an insoluble product. As soon as the product deposits on the surface, the LSPR color changes. After 2 min of reaction with
an estimated 2 nm of product deposited on the surface, the color of the surface becomes blue and corresponds to a shift of
50–100 nm with respect to the original plasmon. B) The proof that color change is specific to a LSPR surface becomes evident
based on the comparison of LSPR and regular gold sensors for the detection of various amounts of alkaline phospahatase. Although
LSPR sensors show a significant dose-response, the regular gold sensor shows minimal response. C) Use of label-free and labeled
LSPR for the detection of residual Protein A (Pro A), in a background of 5 mg/mL IgG. In the left panel, residual Pro A is
spiked in a solution of 5 mg/mL IgG; Pro A is detected label-free in real time to ~10 ng/mL or 2 ng/mg-IgG of Pro A; in the
right panel, the sandwich antibody is labeled with alkaline phosphatase, and its plasmon is read. After addition of the substrate
and 2 min of incubation, the LSPR array is read again to screen for a positive response. The graphs on the right represent
a control surface exposed to BSA (lacking exposure to Pro A) and a surface exposed to 10 pg/mL or 2 pg/mg of IgG of Pro A.
Spectroscopically, the positive surface displays a 44 nm shift, whereas the control shifts by only 6 nm.
The color shift is caused by insoluble substrate being deposited onto the sensor surface, whereby the nanostructured surface
acts as a transducer. The presence, not the color, of the precipitate is measured. For example, the reduction rate of nitro-blue
tetrazolium (NBT) by alkaline phosphatase (0.002–0.007 OD/min)11 corresponds to a deposit on the surface of 2–5 nm/min. On the other hand, the LSPR surface can detect <0.1 nm NBT deposition,
because that amount would cause a measurable index of refraction change at the surface of ~10–3.8 This is why detection is faster than the traditional ELISA when the same type of experiment is performed on the LSPR surface.
The particularities of the nanostructured LSPR surface are illustrated in Figure 3B. Antigen and alkaline phosphatase have
been immobilized in parallel on both the LSPR and regular gold surfaces. After 2 min of substrate conversion, there is a minimal
change on the regular gold chip at an even higher antigen dose, while clear and distinct coloration appears on the LSPR chip.
A real case scenario for the detection of residual Protein A, in a 5 mg/mL IgG background, was undertaken to demonstrate the
feasibility of this approach. Although the label-free detection can detect down to ~10 ng/mL of Protein A in a solution with
5 mg/mL IgG (Figure 3C, left), the labeled method was able to detect down to ~10 pg/mL of Protein A. The labeled-approach
currently is a semiquantitative end-point assay, but there are indications that it can be recast into a real time quantitative
assay as the technology advances.
Monitoring through LSPR requires only a white light source and a spectrometer. The platform also is largely insensitive to
temperature and ultra-precise alignment and therefore can be assembled at a fraction of the cost of conventional SPR. Because
of its engineering simplicity, LSPR is amenable to miniaturization for certain point-of-use assays. Academic laboratories
already have reported palm-size prototypes based on LSPR.12 The emergence of a commercial LSPR platform that reads the absorption of white light by a nanostructured thin film holds
great promise for basic protein science and for applications such as quality control and quality assurance. It permits label-free
quantitation of biomolecules at the ng/mL level, yet makes contaminant detection in solution at femtogram to picogram levels
possible, using the addition of labels that provide notable improvements over current ELISA testing by enhancing the sensitivity
and speed of assay responses. LSPR technology is equivalent to current state-of-the art SPR instruments in terms of data quality,
sensitivity, and dynamic range, while also being scalable relative to throughput. In its current inception, the LSPR instrument
can read eight channels simultaneously with future modules enabling a greater number of channels.
Daniele Gerion and Gwo-Jen Day are both senior scientists at LamdaGen Corporation, Menlo Park, CA, 650.235.5971, firstname.lastname@example.org