4.3. Enzyme-linked immunosorbent assay (ELISA)

ELISA is one of the most renowned techniques that enjoy a high versatility that allowed its use in a variety of biomedical applications including the detection of ANAs and anti-DNA antibodies in patients with SLE and other autoimmune diseases [102–107]. ELISA is based on coating a solid surface with the antigens of interest such as DNA substrates or an array of nuclear antigens so that they are tightly bound and can withstand subsequent washing steps. After immobilization, the test sample containing autoantibodies such as a patient's serum is added to promote binding of autoantibodies with their respective antigens, and the formed immune complexes can be detected through the addition of an anti-IgG antibody that is either conjugated with a peroxidase or alkaline phosphatase enzyme or a fluorophore [44, 94].

ELISA has high sensitivity and can detect a wide spectrum of antibodies owing to the efficient exposure of surface-immobilized DNA substrates making them readily available in high concentrations for binding and can thus capture both high- and low-affinity antibodies [27, 44, 94]. Therefore, ELISA can be a good choice for initial screening [27]. Moreover, it is easy to perform and allows quick, quantitative, and high-throughput analysis of autoantibodies [44]. However, ELISA mediates the recovery of low-affinity anti-DNA antibodies which compromises its specificity to SLE [54]. This can be due to a variety of reasons that can relate to the reaction conditions and the structure and source of the DNA substrate [54]. In addition, the increased sensitivity of ELISA leads to the generation of false-positive results due to cross-reactivity [27, 44].

## 4.4. Farr radioimmunoassay assay

Farr assay has been first introduced in 1968 for the detection of anti-DNA antibodies [108], and until now it is preferred by many clinicians for assaying anti-DNA antibodies as it mediates the selective recovery of high-affinity antibodies which have been described to be reflective of mature immune responses [27]. The principle of the Farr assay is based on the incubation of a solution of radiolabeled DNA such as 14C-DNA with patient's serum sample to promote the formation of immune complexes between anti-DNA antibodies and the DNA substrate. After incubation, immune complexes are precipitated with a saturated solution of ammonium sulfate, and the fraction of the initial radiolabeled DNA that has precipitated with autoantibodies is used to indicate the amount of anti-DNA antibodies in the serum sample [109]. With the ability to only recover high-affinity antibodies, Farr assay exhibits high specificity for SLE, but consequently its sensitivity is not the best when compared with other assays such as ELISA that is able to detect both high- and low-affinity antibodies [27]. In addition, because of the hazardous radioactive material used in DNA labeling and the troublesome associated with its disposal, researches aimed at the development and optimization of other assays including ELISA which at the time suffered inconsistencies with the results owing the lack of standardization protocols with antigen immobilization [93]. However, as will be discussed below, ELISA has been eventually optimized and became one of the popular biomedical techniques in assaying autoantibodies [44].
