4. Diagnostic technologies

Abnormally elevated titers of antinuclear antibodies are one of the important factors involved in SLE pathogenesis [4] and are set as stand-alone criterion in both ACR and SLICC classification criteria used for SLE diagnosis [48, 50]. Out of these antinuclear antibodies, antibodies that target double-stranded DNA are highly specific to SLE and are considered the serological hallmark for SLE [27]. Numerous technologies have been described for the detection of such autoantibodies in the sera, plasma, or other body fluids of SLE patients where some of them date back to the 1950s, and until now various state-of-the-art technologies are being described.

In principle, all assays assess the formation of immune complexes between the autoantibodies and the test antigens which can comprise isolated DNA, complex nuclear mixtures, or an array of purified, recombinant, or synthetic proteins or peptides [27, 44]. These assays, however, differ in many factors including the source of the antigenic substrate, the presentation of such substrates to the autoantibodies where they could be immobilized on solid surfaces or move freely in solution, the methodology encountered for detecting binding and the employed reaction conditions [27, 54]. The variations among the detection assays happened to give conflicting results for the same patient sample where one assay gave positive detection and another had it negative [94–96]. The most important causation of such result variability amongst the different assays is the affinity of autoantibodies towards the test antigen which is highly influenced by the reaction conditions. For example, some assays, as will be described shortly, favor the recovery of low-affinity antibodies such as ELISA, while others assays favor the recovery of high-affinity antibodies such as the Farr assay [27, 97].

Another important aspect with regards to the conflicting results obtained from the distinct assays is the diffidence in the cutoff values set by different assay for considering autoantibody levels detected significant making data from different assays difficult to compare [27]. This confusion actually roots from the vague definition of antinuclear or anti-DNA antibody positivity described in SLICC [50]. The criteria state that an anti-DNA antibody test result is considered positive if it is higher than the laboratory's reference range except for ELISA in which a test result is only positive if it is two times or more the value of the laboratory reference range [27]. That said, the criteria did not define specific tests and did not refer to accepted levels for assay sensitivity or specificity [50, 98].

These discrepancies add to the confusion of diagnosticians who already face many challenges with SLE diagnosis and urge the need for standardization among the different assays. Simplicity, time, and costs are important factors for an assay to be chosen for routine clinical use in laboratories [27].

Amid all the available assays and newly developed technologies that are described for detecting anti-DNA antibodies as the most prominent biomarkers for SLE, an important question is issued of which assay to choose and is there a gold standard to refer to? It has been reported that assays that can specifically detect high-affinity antibodies as being reflective of mature immune responses are preferred by many investigators such as the Farr assay or CLIFT [27, 54]. However, the contribution of high-affinity and low-affinity anti-DNA antibodies to SLE pathogenesis is not yet known, and therefore looking for other biomarkers that can arise from the multiple pathways contributing to SLE pathogenicity is highly needed to complement the value of anti-DNA antibodies in diagnosis and prognosis of SLE [27, 54].

In this section, the most prominent techniques employed for the detection of antinuclear antibodies and anti-DNA antibodies that have been described decades ago till today will be presented, in addition to some investment on the futuristic highly promising state-of-the-art technologies. However, before proceeding with the discussion of the different assays, a description of the DNA substrate antigenic properties and the factors that influence such antigenicity will be discussed as they are important dynamics in assaying anti-DNA antibodies.
