Multiplexed method to calibrate and quantitate fluorescence signal for allergen-specific IgE

Abstract

Using a microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of serum. However, variation of probe immobilization on microarrays hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics. To address this problem, we have developed a calibrated, inexpensive, multiplexed, and rapid protein microarray method that directly correlates surface probe density to captured labeled secondary antibody in clinical samples. We have identified three major technological advantages of our calibrated fluorescence enhancement (CaFE) technique: (i) a significant increase in fluorescence emission over a broad range of fluorophores on a layered substrate optimized specifically for fluorescence; (ii) a method to perform label-free quantification of the probes in each spot while maintaining fluorescence enhancement for a particular fluorophore; and (iii) a calibrated, quantitative technique that combines fluorescence and label-free modalities to accurately measure probe density and bound target for a variety of antibody-antigen pairs. In this paper, we establish the effectiveness of the CaFE method by presenting the strong linear dependence of the amount of bound protein to the resulting fluorescence signal of secondary antibody for IgG, β-lactoglobulin, and allergen-specific IgEs to Ara h 1 (peanut major allergen) and Phl p 1 (timothy grass major allergen) in human serum.

Figure 1

(a) Simulations model the theoretical fluorescence of 100 nm oxide, 320 nm oxide, and glass at normal incidence as a function of emission wavelengths. From left to right, the emission spectra of commonly used fluorophores (Alexa Fluor 359, Alexa Fluor 488, Cy3 dye, Cy5 dye, and Alexa Fluor 647). (b) Fluorescence emission enhancement of Cy3 for 100 and 320 nm oxide is 2-fold compared to glass at NA= 0.7. (c) Normalized reflectivity curves for 500 nm oxide and 320 nm oxide are fitted to illumination wavelengths used in IRIS. (d) The CaFE method is performed and compared for both chip platforms.

Figure 2

(a) Label-free IRIS image of varying concentrations of rabbit IgG on a 500 nm island of Platform-1(CaFE) chip. (b) Fluorescence image of spotting array on CaFE chip for varying concentrations of captured Cy3-antirabbit IgG. CaFE measurement for rabbit IgG (c) and β-lactoglobulin (d). Error bars represent the SD of 20 spots. For IgG calibration of Chip 3, two quadrants (Q1 and Q2) analyze on chip variability. Results show that the CaFE platform yields calibrated, linear responses from chip-to-chip for a variety of proteins.

Figure 3

(a) The standard “calibration” of secondary antibody for diagnosis of allergy is measured as the degree of fluorescence as a function of spotting concentration is shown for Ara h 1 peanut allergen (left) and Phl p1 timothy grass allergen (right) for two chips. Using the self-calibration method provided by CaFE (b), results show calibrated, linear responses for allergy testing analysis compared to traditional “semi-quantifiable” analysis in part a.