Sandwich ELISAs for the detection of anthrax PA with one monoclonal antibody and two polyclonal antibodies have been reported previously (15
). In these systems, the capture antibody was mouse monoclonal antibody M18 or 14B7. These antibodies have specificities similar to the specificity of monoclonal antibody W1 used in our study and are single-epitope capture reagents that target PA domain 4 (4
). Interestingly, these assays, developed independently, were able to achieve the same lower detection limit of ~1 ng/ml for the purified PA protein (15
). However, when Eu+
NPs were used to replace the traditional colorimetric development reagents in the conventional ELISA, the lower limit of detection was dramatically improved by almost 100-fold. Similar results have also been observed for the detection of the B. anthracis
LF protein and the Yersinia pestis
LcrV and F1 proteins, as well as human immunodeficiency virus type 1 p24 antigen (unpublished data). For example, our preliminary data indicate that the current assay conditions can detect 10 pg/ml of anthrax LF by using monoclonal anti-LF antibody and 20 ng/ml of anthrax EF by using PA63 as the capture reagent (data not shown). These results further support the importance of detection chemistries and new labeling technologies in improving assay performance and clearly indicate the potential of NP-based testing methods to enhance the sensitivity of immunoassays and to allow the ultrasensitive detection of pathogens.
Labeling technologies commonly used in immunoassays include radioactivity, enzyme activity, and chemicals (chemiluminescence and fluorescence). The use of radioisotopic labeling is being discontinued in many laboratories for several reasons, including the safety hazards that they pose. One major disadvantage of the enzyme-based colorimetric ELISA is its relatively low detection sensitivity. In the past decade, lanthanide chelates have successfully been used in immunoassays, such as the DELFIA technology. The DELFIA technology is based on the dissociation of lanthanide ions from chelates conjugated to detecting molecules such as SA. This technique yields a high sensitivity with a stable signal, a high signal-to-noise ratio, and a nonenzymatic, flexible platform with a short incubation time. Fortunately, the highly fluorescent chelates used in the DELFIA technique can also be used in fluorescent lanthanide chelate NPs. For example, Harma et al. reported that an Eu+
NP-based immunoassay can detect as little as 1.6 pg/ml of PSA in microtiter wells in a rapid assay (5
). This assay is 100-fold more sensitive than a similar assay that uses conventional Eu+
-labeled SA (5
) and could detect even 0.38 pg/ml of PSA when the analyte was directly biotinylated (6
). Soukka et al. reported an extremely low limit of detection of 0.04 pg/ml for PSA with the Eu+
NP label technology in a two-step immunoassay and a 3-h assay time (27
). The improved sensitivity is due to the high specific activity of the SA-coated NPs, its low nonspecific binding, and the high affinity of the NPs toward the analyte. This is not surprising, given the large surface area of NPs, the capacity to coat a large number of molecules and the huge content of Eu+
ions in a single NP, and in particular, the unique characteristic that the time-resolved lanthanide chelates do not self-quench even when they are used at high millimolar concentrations (5
). In addition, Huhtinen et al. reported that with the use of SA-coated NPs and biotinylated antibody, the steric hindrance caused by the antibody-coated NPs in immunometric sandwich assays can be resolved when the analyte is structurally complex (9
). These unique characteristics indicate that Eu+
NPs, especially SA-coated NPs, may be more useful than Eu+
chelates in the development of assays with high amplification ratios and extremely high sensitivities.
In our study, we also used biotinylated anti-SA antibody and SA-coated Eu+
chelates, followed by the addition of chelating enhancement solution to further enhance the signal intensity, since each NP contains more than 700 SA molecules and more than 2,800 biotin binding sites (5
). We found that the enhancement step could further improve the signal intensity and could increase the sensitivity of the assay by twofold. The major advantage of our current assay format is that all the reagents are universal and can easily be adapted for the detection of different targets by replacing the coating and detection ligands. Furthermore, since the anti-PA IgG antibody is an important marker for the evaluation of anthrax vaccines, anti-PA assays with improved sensitivities would be useful in vaccine development and evaluation. Current assays are able to reach sensitivity limits of 1.5 to 3 μg/ml of anti-PA IgG antibody (2
). We believe that the detection limits for the anti-PA IgG antibody could be further decreased by employing the ENIA. Although our current assay involves several incubation steps, it could be further developed as a two-step assay by using NP-antibody bioconjugate (26
). The entire assay could be completed within 30 min. Therefore, the ENIA format could be suitable for rapid and point-of-care use.
As a proof-of-concept study, our preliminary results indicate that the ENIA described here has the potential to significantly improve the sensitivity of detection of anthrax toxin. The current ENIA also detected purified anthrax toxin and toxin-injected animal samples. The assay could also detect anthrax toxin after infection with B. anthracis
spores, but only after the animals were sick and not at earlier stages of disease, before symptoms were apparent. Similar results have been reported by Mabry et al. (15
). They used an engineered ELISA to measure the amounts of PA in blood samples from guinea pigs and rabbits exposed to a lethal dose of anthrax spores and found that PA could be detected only in the late stages of infection, especially within 12 h of death for the infected guinea pigs or 48 h for the infected rabbits (15
). These findings are not surprising, as the anthrax toxin that is released into the circulation in the early stages of disease is likely to continuously bind to the available tissue receptors until receptor saturation is achieved. Only after saturation will the toxins accumulate in the blood and provide measurable levels in the blood, and the animals are usually sick by this stage.
More studies are needed to develop this nanotechnology-based testing method as a clinical diagnostic assay. Ideally, the early detection of the toxin in infected samples needs to be improved. Improvement of the sensitivity of the assay, coupled with knowledge of the kinetics of infection, may allow the detection of toxin in different sample types, such as infected tissues or cells which bind large amounts of toxin early in infection. In addition, to increase the sensitivity of detection of PA with mutations that may not be detectable with a single monoclonal antibody, a polyclonal anti-PA antibody or a mixture of monoclonal anti-PA antibodies against different PA-reactive epitopes may be used.
Due to the high specificity of SA-coated NPs, the high affinity between biotin and SA, and this universal labeling technology, the assay that we have developed may be suitable for the detection of biotinylated molecules and could be further developed as a rapid and universal testing platform for clinical diagnosis or laboratory research and for resource-limited settings upon further optimization and simplification.