Diagnostic in vitro tests. Immunoassays and cellular tests

The appropriate test for diagnosing allergic diseases depends on the suspected mechanism involved: specific immunoglobulin E (sIgE)-mediated or T cell-mediated, especially in DHRs.22,32-36
IgE-mediated allergic reactions can be induced by aeroallergens, food allergens, and drugs.32,37 Additionally, for the latter, the drug structure coupled to a carrier protein of sufficient size may be involved in the sIgE recognition.38,39 The major issue is the low blood concentration of sIgE, which is approximately 25% of total IgE for aeroallergens; and even lower for drug-sIgE (0.2% for betalactam).40 Thus, extremely high sensitive methods are required.
The best validated and used in vitro approaches are based on the quantification of sIgE, either in serum by immunoassays (radioimmunoassay, enzyme-linked immunosorbent assay, or fluorescent enzyme immunoassay) or on basophil surface by functional basophil activation test (BAT).41-44 The latter is quite specific, but complex to perform, and therefore limited to research laboratories.
Serum sIgE assays against allergen sources/molecules are the most commonly used and can be performed by singleplexed (which use single allergens) or multiplexed strategy.37 In general, they are sensitive but show low specificity due to potential antigenic competition and isotype (IgG) inhibition.45 There are several market leaders of singleplexed assays, whose main advantages are the automation, with increased precision and shorter turnaround times; the miniaturisation chip technology that reduces serum volumes, and the adaptability for use with purified native and recombinant allergens.45 Multiplexed arrays offer the advantage of providing information on the sensitisation pattern of a patient for a large number of molecules with a small amount of serum. However, it can be difficult to differentiate clinically relevant from irrelevant sensitisations.46 Moreover, allergen specificities on multiallergen screen are not defined and differ among various manufacturers.45 Currently, the available multiplex platforms can provide up to 112 allergens (allergen sources and protein groups).46 However, the clinical relevance of many of these epitopes is not known and there is a higher degree of variability in low IgE levels,47,48 cases in which singleplex platforms may be more sensitive.37
In the case of DHRs, solid-phase immunoassays have to include drug-carrier conjugates to detect serum-drug-sIgE.42,49 Due to the extremely low levels of drug-sIgE, they generally have low sensitivity, although this depends on the clinical manifestations, the drug involved, and the time interval between reaction and diagnostic assay.50,51 The carrier molecule can also affect the sensitivity, poly-L-lysine is the most used artificial carrier due to its multivalency, which allows a high hapten density,52 although its polydispersity impedes adequate characterisation, reproducibility, and conjugate control.53 Moreover nonspecific interactions and immobilisation on solid-phase can reduce immunological capture. Both commercial and in-house radioimmunoassays are used, although enhanced sensitivity is needed.54
The use of BAT has increased in the last years, being seen in the overall context of molecular diagnostics in food and aeroallergen allergy.37,43,55 A major issue is the allergen source,37 since results differ according to the variety employed.
In the case of DHRs, BAT has overcome the immunoassay limitations of amount of drugs available and has been mainly studied for neuromuscular blocking agents, betalactams, and iodinated contrast media, with a sensitivity ranging between 50% and 60%, and a specificity of 80%.32 Moreover, BAT has a complementary role for skin test for many drugs to which no other approaches are available.39,56-60 Both commercial BAT and in-house protocols are rarely thoroughly validated and require additional investigation before they can enter mainstream application.37,41

Nanotechnology in biomedicine

The famous conference of Richard Feynman in 1959 and the mythical phrase “there is plenty of room at the bottom” is considered as the starting point of nanotechnology. Nanotechnology is the development of materials with nanometric size for searching new properties at this scale, which could be used for different applications. Some of these materials have been considered as ideal platforms for their functionalisation with ligands with applications in biomedicine.61
Metallic and non-metallic nanoparticles (NPs), carbon nanoforms such as single- or multiple-walls carbon nanotubes (CNTs) and graphene, liposomes, polymers, dendrimers, nanogels, etc. are popular representatives (Figure 1). They show different physical and chemical properties that depend mainly on the size, shape, composition, and functionalisation of the system. Nanomaterial engineering provides the tools to control all these parameters and to achieve the desired requirements for health applications.62 In fact, the functionalisation of these scaffolds is the way to modulate their physical, chemical, and biological properties at will. Their applications in biomedicine include therapeutics, diagnostics, and theranostics 63,64 for drug delivery,65 bioimaging and biosensing,66 as implants,67 cancer immunotherapy,68 gene therapy,69etc. Besides their potential and the expectative in the biomedical area, nowadays not many nanodrugs are approved for medical use, although some promising compounds are still under clinical trials.70Issues related to their nanotoxicity, reproducibility, and homogeneity have impeded a rapid development of this field in health applications.71
There is a plethora of examples, in which these nanomaterials have demonstrated very promising and interesting properties at in vitro level, for which they were decorated with ligands for specific receptors expressed or over-expressed in target cells or tissues. On one hand, these nanosystems behave as selective drugs carriers reducing the toxicity, being selective for specific targets and decreasing side effects. On the other hand, nanostructures can provide the means to protect their cargo, improving their stability against degradation, their solubility, and their drug availability.