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Multiplex Analysis of Autoantibodies

Detecting autoantibodies is critical in various research areas, such as cytokine research, infectious disease research, autoimmune disease research, and cancer research. Multiplex analysis of autoantibodies, such as MILLIPLEX® multiplex assays, helps advance this research by detecting multiple analytes at once to give researchers a full autoantibody profile.

What Are Autoantibodies?

Autoantibodies are antibodies that react with substances/antigens that are self-produced. This happens when the body cannot tell the difference between its own antigens and invading antigens. These autoantibodies lead to inflammation and the destruction of tissues and organs. They are often associated with autoimmune disorders but can be found in other diseases as well.

Examples of autoantibodies include:

  • Cytokine autoantibodies
  • Autoimmune autoantibodies
  • Cancer autoantibodies

Multiplexing Autoantibodies

Multiplex analysis of autoantibodies helps researchers save time in the lab by measuring multiple autoantibodies in one sample. With high-quality multiplexing like MILLIPLEX® multiplex panels using the Luminex® platform, researchers have the option of analyzing a variety of autoantibodies as well as the flexibility to choose which analytes they want to assay. Figure 1 shows the capture antigen method these assays use.

MILLIPLEX® autoantibody kits are for qualitative measurement of autoantibodies in serum and plasma samples. Each kit contains four assay controls beads to be mixed with the selected antigen-immobilized beads and a PE-Ig conjugate as the detection component.

Depiction of the MILLIPLEX® autoantibody panel assay method. This represents a capture antigen method, with the Luminex® magnetic bead, capture antigen, IgG autoantibody (analyte), and phycoerythrin-anti-human IgG conjugate.


Figure 1.
The MILLIPLEX® autoantibody panel assay depiction. This represents a capture antigen method.

Measuring Cytokine Autoantibodies

Measuring cytokine autoantibodies may be useful in disease research since anti-cytokine antibodies occur frequently and are present in healthy and patient samples with various infections, acquired immunodeficiency, or autoimmune diseases. Cytokines offering protection against microbes can be targeted by cytokine autoantibodies, leading to life-threatening infections. Measuring cytokine autoantibodies may be useful for research on disease monitoring and the efficacy of treatment.

Cytokine Autoantibody Multiplex Analysis

With multiplex analysis of cytokine autoantibodies, you can measure more biomarkers in one sample. Our MILLIPLEX® Human Cytokine Autoantibody Panel contains antigen-coated beads for the simultaneous detection of 15 autoantibodies in serum or plasma samples. Four assay control beads are included as part of the base kit, and you can select the cytokine autoantibodies you want to assay as a part of your experiment. Figure 2 shows example data of using this panel in autoimmune disease and sepsis samples, compared to healthy samples.

Graph showing data for anti-TNFα, anti-IL-8, anti-IL-18, and anti-IL-15 analysis. Using MILLIPLEX® Human Cytokine Autoantibody Panel Cat. No. HCYTAAB-17K, the MFI (Median Fluorescence Intensity) values of the cytokine autoantibody biomarkers were determined in autoimmune disease, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) (n=19), sepsis (n=17), and healthy (n=9) serum samples (diluted 1:100). Data from 4 select analytes in the kit are shown.
Graph showing data for anti-TNFα, anti-IL-8, anti-IL-18, and anti-IL-15 analysis. Using MILLIPLEX® Human Cytokine Autoantibody Panel Cat. No. HCYTAAB-17K, the MFI (Median Fluorescence Intensity) values of the cytokine autoantibody biomarkers were determined in autoimmune disease, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) (n=19), sepsis (n=17), and healthy (n=9) serum samples (diluted 1:100). Data from 4 select analytes in the kit are shown.

Figure 2.Using Product Number HCYTAAB-17K, the MFI (Median Fluorescence Intensity) values of the cytokine autoantibody biomarkers were determined in autoimmune disease, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) (n=19), sepsis (n=17), and healthy (n=9) serum samples (diluted 1:100). Data from 4 select analytes in the kit are shown.


Autoimmune Autoantibody Analysis

Autoantibody formation is principal to the pathogenesis of a variety of autoimmune diseases. Dysregulated apoptosis and the subsequent defective clearance of cellular debris lead to the exposure of autoantigens and the generation of autoantibodies. The presence of autoantibodies may indicate disease activity, prognosis, and clinical associations related to a variety of autoimmune diseases, including systemic lupus erythematosus (SLE), Sjögren’s Syndrome, Systemic Sclerosis, Polymyositis (PM)/Dermatomyositis, and various overlap syndromes of these diseases. Table 1 describes autoimmune autoantibodies and their disease associations.


Table 1.
Autoimmune autoantibodies and their disease associations. Abbreviations: MCTD – Mixed connective tissue disease, SCLE - Subacute cutaneous lupus erythematosus, LE - lupus erythematosus, CNS – central nervous system, CREST - calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia.

Autoimmune Autoantibody Multiplex Analysis

The MILLIPLEX® Human Autoimmune Autoantibody Panel contains 20 antigens for detection in serum or plasma samples. These autoantibodies include:

  • β2-Glycoprotein
  • C1q
  • CENP-A (Centromere Protein A)
  • CENP-B (Centromere Protein B)
  • Jo-1
  • Ku
  • Mi-2
  • Myeloperoxidase (MPO)
  • PCNA (Proliferating Cell Nuclear Antigen A)
  • PL-12 (Alanyl-tRNA synthetase)
  • PM/Scl 100
  • Proteinase 3
  • Ribosomal P
  • RNP (Ribonucleoprotein)
  • RNP/Smith (RNP/Sm)
  • Scl-70
  • Smith (Sm)
  • SSA/Ro52 (Sjögren's Syndrome-related antigen A/Ro52 kDa)
  • SSA/Ro60 (Sjögren's Syndrome-related antigen A/Ro60 kDa)
  • SSB/La (Sjögren's Syndrome-related antigen B/La)

Figure 3 shows a comparison of disease and normal samples using this panel.

Graph showing data for autoimmune autoantibody analysis. Using MILLIPLEX® Human Autoimmune Autoantibody Panel Cat. No. HAIAB-10K, the Median Fluorescence Intensity (MFI) were determined for 19 disease samples – 10 systemic lupus erythematosus, 7 granulomatosis with polyangiitis, and 2 polymyositis, and 10 normal samples. Disease samples were much higher for 14 of the 20 analytes in this panel.

Figure 3.Using Product Number HAIAB-10K, the Median Fluorescence Intensity (MFI) was determined for 19 disease samples – 10 systemic lupus erythematosus, 7 granulomatosis with polyangiitis, and 2 polymyositis, and 10 normal samples. Disease samples were much higher for 14 of the 20 analytes in this panel.

Figure 4 shows autoantibody MFI values compared to clinical laboratory data, showing that this panel followed similar trends to what was measured by a commercial lab.

Graph showing data for autoimmune autoantibody analysis of MILLIPLEX® MFI related to commercial lab results. Clinical laboratory data were available for 6 autoantibodies (SSA/Ro60, SSA/Ro52, SSB/La, RNP, Sm, and Scl-70) in 8 SLE patients. Those SLE patients positive by clinical laboratory methods also demonstrated elevated MFI in the MILLIPLEX® Autoimmune Autoantibody panel. The SLE patients considered negative by the same clinical laboratory methods exhibited lower MFI.

Figure 4.Clinical laboratory data were available for 6 autoantibodies (SSA/Ro60, SSA/Ro52, SSB/La, RNP, Sm, and Scl-70) in 8 SLE patients. Those SLE patients positive by clinical laboratory methods also demonstrated elevated MFI in the Autoimmune Autoantibody panel. The SLE patients considered negative by the same clinical laboratory methods exhibited lower MFI.

Figure 5 describes the reference serum analysis with this panel.

Graph showing data for autoimmune autoantibody analysis. Reference sera were available for evaluation of 10 autoantibodies in the MILLIPLEX® Human Autoimmune Autoantibody Panel. The controls exhibited elevated MFI for the autoantibodies for which they were expected to react strongly.
Graph showing data for autoimmune autoantibody analysis. Reference sera were available for evaluation of 10 autoantibodies in the MILLIPLEX® Human Autoimmune Autoantibody Panel. The controls exhibited elevated MFI for the autoantibodies for which they were expected to react strongly.
Graph showing data for autoimmune autoantibody analysis. Reference sera were available for evaluation of 10 autoantibodies in the MILLIPLEX® Human Autoimmune Autoantibody Panel. The controls exhibited elevated MFI for the autoantibodies for which they were expected to react strongly.

Figure 5.Reference sera were available for evaluation of 10 autoantibodies in the MILLIPLEX® Human Autoimmune Autoantibody Panel. The controls exhibited elevated MFI for the autoantibodies for which they were expected to react strongly.

This shows the value of using multiplex technology to evaluate multiple autoantibodies, allowing for an extensive autoantibody profile to evaluate samples from various autoimmune diseases.


Cancer Autoantibody Detection

Cancer autoantibodies to tumor-associated antigens are being studied as potential cancer biomarkers, as well as possible reporters of early carcinogenesis and indicators of cancer prognosis. Research is being done to find accurate, reliable, and affordable blood tests that are needed for the early detection of cancer.

Cancer Autoantibody Multiplex Analysis

The MILLIPLEX® Human Cancer Autoantibody Panel contains cancer antigen-coated beads for simultaneous detection of 15 autoantibodies in serum or plasma samples. It offers the ability to select the cancer autoantibodies needed for your experiment. Figure 6 shows a comparison between cancer and normal samples using this panel.

Graph showing data for cancer autoantibody analysis. Using MILLIPLEX® Human Cancer Autoantibody Panel Cat. No. HCABMAG-13K, the MFI (Median Fluorescence Intensity) values of the cancer autoantibody biomarkers were determined in cancer (breast, colorectal, ovarian, lung, prostate) (n=78) and healthy control (n=18) serum samples (samples diluted at 1:100).
Graph showing data for cancer autoantibody analysis. Using MILLIPLEX® Human Cancer Autoantibody Panel Cat. No. HCABMAG-13K, the MFI (Median Fluorescence Intensity) values of the cancer autoantibody biomarkers were determined in cancer (breast, colorectal, ovarian, lung, prostate) (n=78) and healthy control (n=18) serum samples (samples diluted at 1:100).
Graph showing data for cancer autoantibody analysis. Using MILLIPLEX® Human Cancer Autoantibody Panel Cat. No. HCABMAG-13K, the MFI (Median Fluorescence Intensity) values of the cancer autoantibody biomarkers were determined in cancer (breast, colorectal, ovarian, lung, prostate) (n=78) and healthy control (n=18) serum samples (samples diluted at 1:100).
Graph showing data for cancer autoantibody analysis. Using MILLIPLEX® Human Cancer Autoantibody Panel Cat. No. HCABMAG-13K, the MFI (Median Fluorescence Intensity) values of the cancer autoantibody biomarkers were determined in cancer (breast, colorectal, ovarian, lung, prostate) (n=78) and healthy control (n=18) serum samples (samples diluted at 1:100).

Figure 6.Using Product Number HCABMAG-13K, the MFI (Median Fluorescence Intensity) values of the cancer autoantibody biomarkers were determined in cancer (breast, colorectal, ovarian, lung, prostate) (n=78) and healthy control (n=18) serum samples (samples diluted at 1:100).

Figure 7 describes the reactivity of serial diluted serum cancer autoantibody samples.

Graph showing data for reactivity of serial diluted serum cancer autoantibody samples. The MILLIPLEX® cancer autoantibody multiplex assay is robust and requires less than 5 µL of human serum samples, as the sera were tested at 1:100 dilution. Note twelve of the fifteen analytes are shown.

Figure 7.The cancer autoantibody multiplex assay is robust and requires less than 5 µL of human serum samples, as the sera were tested at 1:100 dilution. Note twelve of the fifteen analytes are shown.

This human cancer autoantibody multiplex assay can be used alone or in combination with other MILLIPLEX® cancer circulating biomarker panels, as useful biomarker screening tools in studying the early detection of cancer.


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