4.7.2. Addressable laser bead immunoassays

Addressable laser bead immunoassays (Figure 4) [112] are based on coupling distinct antigens (up to 100) onto microbeads that come in multiple laser reactive colors where each antigen is coupled to a specific color of beads creating an "addressable" color code that is used for the identification of each antigen [93]. After antigen coupling, all beads are stabilized, collected into a microtiter well, and incubated with the sample, and the formed immune complexes are detected by a fluorescently tagged secondary antibody. Samples are then analyzed using a dual laser system that utilizes flow cytometry and digital signal processing. Each bead is hit with two laser beams where one detects the specific color code or address of the microbead that is used to identify the specific antigen, and the other laser beam detects and quantifies the fluorescence signal coming from the secondary antibody. The generated data provides quantitative and qualitative analysis of each autoantibody [93].

Both LIA and addressable laser bead immunoassays use defined sets of identified antigens which allows the detection of specific autoantibodies associated with a certain disease such as anti-DNA antibodies in SLE which is of high diagnostic value and can thus overcome one of the main limitations of IFA and ELISA in which assigning absolute specificities to an

Figure 4. Addressable laser bead immunoassay (ALBIA). (A) ALBIA involves coupling of each individual antigen onto microbeads of laser active color so that antigens can be "addressable" by the color of the beads. (B) After coupling, all beads are combined into the microtiter well where they are incubated with the test sample such as patient's serum sample to promote formation of immune complexes which are detected by a secondary antibody that is fluorescently labeled. (C) Autoantibody-antigen complexes that are coupled to the beads are analyzed using a dual laser system where one detects the specific color code of the microbead and can thus be used to identify the specific antigen and the other laser beam detects and quantifies the fluorescence signal coming from the bound secondary antibody [112].

ANA-positive result is not possible. Furthermore, these assays are less time-consuming, are amenable for automation, and allow high-throughput analysis. However, this specificity stemming from the use of defined set of antigens can come at the expense of sensitivity where many autoantibodies can be missed. For example, SLE patients can release up to 200 different autoantibodies where only few of them are detected [113].

### 4.7.3. Autoantigen microarrays

Autoantigen microarrays are high-throughput assays that allow simultaneous detection of different autoantibodies in autoimmune diseases including SLE (Figure 5) [52]. The technique involves printing a distinct array of antigens into a surface that is coated with substrate that becomes covalently attached to the antigens. Printed antigens on the surface of microarrays are performed in a way that retains their reactivity with other molecules. In SLE, the array of antigens can include nuclear antigens including RNA and DNA and associated proteins, other cellular proteins, and specific targeted epitopes where the immobilized antigens can be purified or recombinant proteins or synthetic peptides. Different samples can be screened including serum, plasma, or other biological fluids such as cerebrospinal fluids or saliva for simultaneous detection of autoantibodies. Identification of immune complexes is mediated through the addition of secondary antihuman antibodies that are fluorescently labeled. Distinct isotypes of

Figure 5. Simultaneous detection of autoantibodies using autoantigen microarrays. (A) Multiple autoantigens are printed onto a microarray surface and are incubated with patients' serum sample that contains autoantibodies. The formed immune complexes are identified through the addition of secondary antibodies that are labeled with a fluorophore so that each spot with a positive reaction produces a fluorescent signal. (B) Different isotypes of autoantibodies such as IgG and IgM can be visualized using secondary anti-IgG and anti-IgM that are labeled with differently colored fluorophores such as Cy5 and Cy3. Merging the images will show the different isotypes in the same sample [52].

autoantibodies such as IgG, IgM, or others can be specified by using secondary antibodies that are conjugated to different colored fluorophores. Autoantigen microarrays are not only useful in showing the differential detection of autoantibodies, but specific associations of certain autoantibodies with certain SLE manifestations can be made in addition to the identification of novel biomarkers. However, microarray chips can be challenging to be produced in consistent and reproducible manner for commercialization.
