Application of photonic crystal enhanced fluorescence to detection of low serum concentrations of human IgE antibodies specific for a purified cat allergen (Fel D1)

Abstract

We demonstrate the detection of low concentrations of allergen-specific Immunoglobulin E (IgE) in human sera using a Photonic Crystal Enhanced Fluorescence (PCEF) microarray platform. The Photonic Crystal (PC) surface, designed to provide optical resonances for the excitation wavelength and emission wavelength of Cy5, was used to amplify the fluorescence signal intensity measured from a multiplexed allergen microarray. Surface-based sandwich immunoassays were used to detect and quantify specific IgE antibodies against a highly purified cat allergen (Fel d1). A comparison of the lowest detectable concentration of IgE measured by the PC microarray system and a commercially available clinical analyzer demonstrated that the PCEF microarray system provides higher sensitivity. The PCEF system was able to detect low concentrations of specific IgE (~0.02 kU/L), which is 5-17-fold more sensitive than the commercially available FDA-approved analyzers. In preliminary experiments using multi-allergen arrays, we demonstrate selective simultaneous detection of IgE antibodies to multiple allergens.

Keywords: Allergen microarray; High sensitivity; Photonic Crystal Enhanced Fluorescence.

Figure 1

(a) Schematic of the Photonic Crystal (PC) structure and the laser scanning detection instrument. The PC is comprised of a periodic surface structure fabricated in a low refractive index (RI) silicon dioxide (SiO₂) layer on a silicon substrate that is overcoated with a thin film of high refractive index TiO₂. (b) An SEM image showing the surface structure of the PC. (c) Schematic diagram of the detection instrument. (d) Reflected intensity as a function of incident angle of the PC when it is illuminated by a Transverse Magnetic (TM) polarized laser at a wavelength of λ=637nm. The peak location of the spectrum indicates the resonance condition is achieved at the incident angle of 4.12°.

Figure 2

Schematic diagram illustrating the major assay steps. A PC surface was first silanized, after which allergens were printed in the form of micro-spots. The PC surface was subsequently blocked to prevent any further protein binding to the surface. Human serum containing IgE was incubated and unbound IgE was removed. Next, biotinylated anti-IgE was added as the detection antibody. Following the wash step, SA-Cy5 was added as the fluorescent tag.

Figure 3

(a) A PC holds 10 subarrays, and each subarray contains 4 sets of 4 replicate spots per protein for a total of 16 spots. Inset: schematic of the microarray layout. The first row is the cat hair extract, the second row is the Fel d1, the third row is the Timothy grass extract and the last row is a set of array location fluorescent spots comprised of Alexa-Fluor-555 fluorescencent streptavidin conjugates. (b) A 10 well format custom-made slide module assembly in which the PC is inserted during the assay steps. (c) Fluorescence images of the arrays tested with different sera. (d) Average fluorescence intensities from Fel d1 and Timothy grass spots. The result indicates that the fel d1 and grass pollen assays exhibited excellent selectivity. Strong fluorescence was observed on grass pollen spots for 4-fold dilution of serum #85228 which had a high concentration of grass-pollen specific antibodies, while no fluorescent signal was observed for 4-fold dilution of serum #924365 that had low concentration. No fluorescence was detected on either grass extract or Fel d1 spots for the negative serum.

Figure 4

Fluorescent images of microspots at different IgE antibody concentrations obtained by dilution of two different serum samples. At each dilution the Feld1 spots have higher fluorescence intensities than the crude cat extract spots, confirming that purified allergen has stronger binding capacity and potentially higher diagnostic sensitivity than crude extracts.

Figure 5

Standard curve for Fel d1 allergen detection. The black solid line in the inset shows the background intensity, which is the blank intensity from the negative control (indicated by the dashed line) plus three times the standard deviation. We consider fluorescence signals above the background intensity as detectable. Therefore, the lowest detectable concentration for Fel d1-specific antibody is ~0.02 kU/L, which is lower than that measured by the ImmunoCAP system.

Figure 6

Representative images of the allergen spots assayed with patient sera at various IgE levels ranging from 0.089 kU/L to 40 kU/L. Note that the PCEF array system is capable of detecting the low concentration of IgE in serum #9, which is not observable using the ImmunoCAP system.