Autoantibodies in LE R3 Anchisa Srivipatana

Antinuclear antibody (ANA)

ANA ANA assay identifies antibodies present in serum that bind to autoantigens present in the nuclei (or cytoplasm) of mammalian cells Many subtypes of ANAs

Autoantibody associations of ACLE ACLE are those associated with SLE high-titer ANA, anti-dsDNA, anti-Sm, and hypocomplementemia

Autoantibody associations of SCLE anti-Ro/SS-A 70%–90% anti-La/SS-B 30%–50% ANA 60%–80% rheumatoid factor 33% false-positive serologic tests for syphilis (VDRL rapid plasma reagin) 7%–33% anticardiolipin 10%–16% antithyroid 18%–44% anti-Sm 10% anti-ds-DNA 10% anti-U1 ribonucleoprotein 10%

Autoantibody associations of DLE ANA are present in low titer in 30%–40% Fewer than 5% have the higher ANA levels that are characteristic of patients with overt SLE (>1:320) Antibodies to ssDNA, dsDNA are uncommon Ro/SS-A and La/SS-B autoantibodies are rare in patients with DLE

ANA Nucleated substrate: Human tumor cell line such as Hep­2 cell Auto Ab are detected with a fluorochrome­ conjugated antiserum that is specific for human immunoglobulin (the aAb) that is bound to nuclei in the cell substrate The use of Hep­2 cells, only ~1–2% of SLE patients are ANA­negative ANA in SLE patients: sensitivity 98% and specificity 90%

ANA test Indirect immunofluoresence (Report pattern and titer) Enzyme-linked immunosorbent assay (ELISA) Crithidia luciliae test

Indirect immunofluorescence One of the most commonly used tests for ANAs Substrate: HEp-2 cell

Stages of immunofluorescence for the detection of antinuclear antibodies (1)HEp-2 cells are permeablised (2)Incubated with a person's blood serum If the serum contains antibodies, they will bind to antigens within the HEp-2 cell nucleus (3) These antibodies can be visualised by subsequent incubation with anti-human antibodies conjugated to a fluorescent molecule

ANA patterns

Immunofluorescence Patterns of ANA Homogenous pattern Peripheral pattern (Rim pattern)

Speckled pattern Nucleolar pattern

Immunofluorescence Patterns of ANA Centomeric pattern (Discrete speckled pattern)

Positive ANA in other AI-CTDs Systemic sclerosis 90% Sjogren’s syndrome 70% Idiopathic inflammatory der­ matomyopathies (DM/PM) 40–65%

Homogeneous and speckled pattern Most laboratories report patterns Commonly occur in healthy and older individual Clinically relevant ; High titer ≥1 : 160

Peripheral pattern Presence of autoAb to DNA, including dsDNA

Centromeric pattern Antibodies binding to the polypeptide components of chromosomal centromeres Associated primarily with the limited form of SSc

Nucleolar pattern Auto Ab binding to ribosomal RNA processing molecules such as fibrillarin

The popular ELISA assays preferentially detect low-avidity anti­ dsDNA aAb that are also seen in patients who do not have SLE. It is recom­ mended that all positive anti­ dsDNA aAb ELISA results be confirmed by a second assay technique that detects high-avidity anti­dsDNA aAb, such as the Farr radioimmunoassay or the Crithidia luciliae indirect immunofluorescence assay.

Crithidia luciliae Crithidia luciliae are haemoflaggellate single celled protozoa Used as a substrate in immunofluorescence for the detection of anti-dsDNA antibodies Organelle known as the kinetoplast which is a large mitochondria with a network of interlocking circular dsDNA molecules

Crithidia luciliae After incubation with serum containing anti-dsDNA antibodies and fluorescent- labelled anti-human antibodies, the kinetoplast will fluoresce. The lack of other nuclear antigens in this organelle means that using C.luciliae as a substrate allows for the specific detection of anti-dsDNA antibodies

Crithidia luciliae

ELISA method

Anti­Ro/SS­A and Anti­La/SS­B aAb

Anti­Ro/SS­A and anti­La/SS­B aAb AutoAb react with proteins in human cytoplasmic RNP (hYRNP) that are found in both the cytoplasm and nucleus “linked set” of autoantigens by present in the same RNP particles.

Anti­RNP aAb assays To measure anti­Ro/SS­A, anti­La/SS­B, anti­U1RNP and anti­Sm aAb Detect method : ELISA ELISA is more sensitive but less specific for detecting anti­RNP aAb

Anti­Ro/SS­A and anti­La/SS­B aAb Anti-Ro antibodies are specific to components of the Ro-RNP complex, comprising 45kDa, 52kDa, 54kDa and 60kDa proteins and RNA The 60kDa DNA/RNA binding protein and 52kDa T-cell regulatory protein are the best characterised antigens of anti- Ro antibodies

Anti­Ro/SS­A and anti­La/SS­B aAb The La antigen is a 48kDa transcription termination factor of RNA polymerase III, which associates with the Ro-RNP complex

Speckled Immunofluoresence staining pattern of anti-nuclear antibodies on HEp cells. This staining pattern is seen with anti-Ro and anti-La antibodies

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