Detection of autoantibodies using chemiluminescence technologies

Figures & data

Figure 1. Concept and protocol of the QUANTA Flash assays. Figure panel (a) illustrates the QUANTA Flash assay cartridge design, which is a key feature making the BIO-FLASH easy to use. Panel (b) shows the overview of QUANTA Flash assay scheme. The procedure involves three reactions (capture of the antibody of interest from the sample by the antigen coupled to the beads; recognition with the ABEI-labelled antibody; chemiluminescent measuring) separated by two washing steps. All steps are performed automatically by BIO-FLASH. Panel (c) illustrates the chemical reaction of the light emission. Using an amide linkage (or pseudo-peptide), the antigen (Ag) or antibody (Ab), indicated as an R in the figure, is attached to the aminobutylethylisoluminol (ABEI) molecule separated by a spacer, therefore reducing the quenching effect of the protein. In the presence of H₂O₂, a catalyst and high pH, light is emitted. The height and width of the peak of light depends upon the quantity of ABEI captured by the bead, which is directly proportional to the concentration of relevant analyte present in the patient sample. The output is measured over 3 s (from second 9 to second 12) and yields a number value in relative light units (RLUs). In (d), an example of Master (dotted) and Working (green solid) curve is illustrated above. The RLUs are expressed on the y-axis and chemiluminescent units (CU) on the x-axis.

Figure 2. Illustration of the high surface capacity of paramagnetic beads in comparison to conventional ELISA. Paramagnetic beads provide a significantly higher surface area compared to ELISA plates and are able to bind more antigen or antibody which often results in high analytical sensitivity and a broad analytical measuring range.

Table 1. Overview of QUANTA flash assays.

No.	Assay	Family	Analytical measuring range (CU)	Comment	Studies (references)
1	CTD Screen Plus	CTD	N/A^a	Similar performance compared to ANA by HEp-2 IIF	14
2	DFS70	CTD	3.2–450.8	Biomarker to differentiate AARD from non-AARD in ANA positive individuals	9^,15^,22
3	ENA7	CTD	3.6–429.4	Excellent correlation with confirmation assays	8
4	dsDNA	CTD	9.8–666.9	Good clinical performance with high specificity for SLE.	47
5	SS-A/Ro60	CTD	4.9–1374.8	Uses new generation of recombinant antigen. Evaluated in clinical comparison to MFI.	8^,16
6	Ro52	CTD	2.3–1685.3	Evaluated in clinical comparison to MFI. Good correlation with ENA7 screen assay.	8^,16
7	SS-B	CTD	3.3–1706.8	Evaluated in clinical comparison to MFI. Good correlation with ENA7 screen assay.	8^,16
8	RNP	CTD	3.5–643.8	Good correlation with ENA7 screen assay.	8
9	Sm	CTD	3.3–693.5	Good correlation with ENA7 screen assay.	8
10	Jo-1	CTD	2.2–1147.2	Good correlation with ENA7 screen assay.	8
11	Scl-70	CTD	3.8–969.8	Good correlation with ENA7 screen assay.	8
12	Centromere	CTD	3.4–708.9	Uses CENP-B antigen	N/A
13	Ribosomal P	CTD	1.6–362.0	Uses synthetic peptide	N/A
14	CCP3	RA	4.6–2776.7	Good clinical performance and correlation with other solid phase methods.	27
15	tTG IgA	CD	1.9–4965.5	Good clinical performance found in CD. High odds ratio at > 10 × ULN	12
16	tTG IgG	CD	3.8–2560.0	Good clinical performance found in CD.	12
17	DGP IgG	CD	2.8–1936.7	Comparable clinical performance to FEIA found in CD	12
18	DGP IgA	CD	5.2–2367.3	Comparable clinical performance to FEIA found in CD	12
19	DGP Screen	CD	0.5–1461.8	N/A	N/A
20	ACL IgG	APS	0.6–2024	Good clinical performance and correlation with other solid phase methods.	32–34
21	ACL IgM	APS	1.0–774.0	Good clinical performance and correlation with other solid phase methods.	32–34
22	ACL IgA	APS	1.4–351.6	Good clinical performance and correlation with other solid phase methods.	32–34
23	B2 IgG	APS	6.1–6400.0	Good clinical performance and correlation with other solid phase methods.	32–34
24	B2 IgM	APS	1.1–841.0	Good clinical performance and correlation with other solid phase methods.	32–34
25	B2 IgA	APS	4.0–512.0	Good clinical performance and correlation with other solid phase methods.	32–34
26	Domain 1	APS	3.6–1380.4	Specific marker for the diagnosis of APS and risk management of APS patients	10^,23^,35
27	PR3	SVV	2.3–3285.3	Good clinical performance found in SVV. Potentially useful in ulcerative colitis and PSC.	13^,20^,25^,26^,38
28	MPO	SVV	3.2(− 739.8	Good clinical performance found in SVV.	38
29	GBM	SVV	2.9–1437.8	Good clinical performance when evaluated in an international multicenter study	29

APS, anti-phospholipid syndrome; CD, celiac disease; CTD, connective tissue disease; FEIA, fluoroenzyme immunoassay; SjS, Sjogren's syndrome; IIM, idiopathic inflammatory myopathies; RA, rheumatoid arthritis; MCTD, mixed connective tissue disease; SVV, small vessel vasculitis; ULN, upper limit of normal; MFI, multiplex fluorescent immunoassay; N/A, not available; SLE, systemic lupus erythematosus; IIF, indirect immunofluorescence; PSC, primary sclerosing cholangitis; ANA, anti-nuclear antibodies.

^aQUANTA Flash CTD Screen Plus is a qualitative assay.

Figure 3. Agreement between QUANTA Flash assays and other autoantibody detection methods according to Cohen’s kappa agreement test. Red error bars indicate 95% confidence intervals (CI), although not available for all published studies. QF, QUANTA Flash; CTD, connective tissue disease; RA, rheumatoid arthritis; CD, celiac disease; APS, anti-phospholipid syndrome; SVV, small vessel vasculitis.

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