4.1. DNA substrate

There are general characteristics of DNA that made it possible to employ DNA isolated from distinct sources including viral, bacterial, mammalian, and even flagella as the antigen substrates in multiple anti-DNA antibody detection assays [27, 54]. For instance, DNA is generally a highly charged polymeric molecule with repetitive charges that constitute an important factor of DNA epitopes that are targeted by anti-DNA antibodies. In addition, all doublestranded DNA isolated from natural origins predominantly exist in the B-form conformation rendering them recognizable by anti-DNA antibodies [27].

That said, there are some factors that can influence DNA antigenicity rendering it more or less antigenic and can thus consequently influence the performance of detection assays [94]. For instance, single-stranded DNA has a much more flexible structure than DNA double helix which tends to be more rigid, and thus targeted epitopes are more exposed in the singlestranded forms making it more antigenic [94]. Another factor that can influence DNA antigenicity is size. Anti-DNA antibodies bind DNA through a mechanism called monogamous bivalency in which both Fab fragments of the same antibody bind the same polynucleotide chain to ensure stable binding [27]. The distance between two Fab sites is 136 angstroms which corresponds to a stretch of 40 bp (Figure 2) [27]. Therefore, short oligonucleotides can be inefficient for binding anti-DNA antibodies, and longer DNA substrates can be essential for binding [27, 94]. Furthermore, certain isolated DNA such as that from Crithidia luciliae protozoal cells display a bent conformation resembling that of nucleosomes allowing binding only a subset of autoantibodies [27].

## 4.2. Immunofluorescence assay (IFA)

IFA (also known as indirect IFA) is an assay that dates back to the 1960s that was developed with the aim of providing sensitive and reliable means for the detection of ANAs [99]. As mentioned earlier, ANA positivity is one of the classification criteria described for SLE diagnosis, and IFA was mentioned by name in the ACR classification for detecting ANA [48]. Moreover, it is considered the gold standard for ANA testing [44, 48, 49, 90]. IFA involves fixation of culture cells on a slide to serve as the source of antigens targeted by ANAs, and

Figure 2. Monogamous bivalency binding of anti-DNA antibodies to DNA. Anti-DNA antibodies bind to the DNA via a mechanism known as monogamous bivalency in which both Fab sites bind the same stretch of polynucleotides. The distance between two Fab sites is 136 angstroms which corresponds to 40 base pairs. Accordingly, DNA fragments of shorter sizes are not efficient as substrates in anti-DNA antibody assays [27].

HEp-2 cells are currently employed for this purpose as they express a wide spectrum of antigens (Figure 3) [44]. Fixed cells are then incubated with serial dilutions of the patient's serum or plasma to promote the formation of immune complexes which is then followed by washing off unbound antibodies. The formed immune complexes are then detected by adding an anti-IgG antibody that is conjugated with a fluorescent agent and are visualized by fluorescence microscopy [44]. Positivity is assigned through determining the endpoint titer which is defined as the reciprocal of the last dilution that gave a fluorescence signal above the cutoff value [44, 100]. The specificity of binding can be inferred from examining the staining pattern which reflects the relative location of the antigen [44].

However, IFA is challenged by certain limitations that can compromise its specificity. First, whole cells are used as the source of test antigens which leads to the detection of other

Figure 3. Immunofluorescence assay (IFA). IFA starts by fixation of culture cells on a glass slide which serves in providing the nuclear antigens that are targeted by ANAs in the patient's serum. Next, different dilutions of the patient's serum are incubated with the cells and are followed by a washing step to remove unbound antibodies. Immune complexes are detected by adding fluorescently labeled anti-IgG antibodies which are followed by a second washing step to remove unbound anti-IgG antibodies. Immune complexes are then visualized and analyzed through fluorescence microscopy [44].

antibodies targeting cytoplasmic and mitotic antigens that complicate the analysis [44]. Furthermore, certain antigens can be underexpressed in these cells which limit the detection of certain autoantibodies [101]. Moreover, IFA execution is subject to variability in experimental conditions including cell fixation protocols, concentrations of assayed cells, different assays of the commercially available kits, and the specificity of the anti-IgG antibodies [44]. Therefore, many efforts have been devoted at formulating other assays that can detect ANAs in a more robust and less technically demanding fashion and capable of high-throughput screening such as enzyme-linked immunosorbent assay (ELISA).
