| ANA/AMA-Immunoblot
or the detection of antinuclear and antimitochondrial antibodies Here you see an ANA/AMA-Immunoblot scanned with the AID Blot Scanning System.
ANA/AMA Cut off: The standard setting is 15 %, can be adjusted if required. The following antibodies can be detected:
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| Comments: The antigen on the ANA/ AMA immunoblot is a nuclear extract from HEp 2 cells (enriched with cytoplasmic proteins). This immunoblot has the advantage of being able to confirm immunofluorescence patterns (except ENA). Only approx. 25 %-30% of these fluorescence patterns are ENAs, the remaining 70% are non-definable bands. |
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| Relevance: The serological characterisation of autoimmune diseases is achieved by the detection of various autoantibodies or typical autoantibody profiles.The majority of these antibodies are directed against nuclear and cytoplasmic antigens of the mitochondria and microsomes. Antinuclear antibodies (ANA) are predominantly associated with rheumatic diseases.Several of these ANAs are disease-specific and can be used as diagnostic markers.These include antibodies against: · Double strand DNS (ds-DNS) and the Sm-antigen in cases of systemic lupus · Topoisomerase-I in cases of diffuse scleroderma · Centromeres (ACA) in cases of CREST · Histidyl-tRNA-synthase (Jo-1) in cases of polymyositis · PM-Scl in cases of overlapping polymyositis and scleroderma Anti-mitochondrial antibodies (AMA),type anti-M2, react with the proteins of the -ketoacid in the hydrogenase complex of the mitochondria. |
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| Clinical
interpretation and notes on the extractable nuclear antigens (ENAs)
detectable in the immunoblot |
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| Anti-AMA
(M2) The detection of anti-AMA M2 has a high clinical sensitivity and a specifity of 97 %. The detection of mitochondrial antibodies type M2 is clinically induced for: · the diagnosis of primary biliary cirrhosis (PBC) and · the differential diagnosis of chronic active hepatitis (CAH) Comments: Anti-AMA (M2) are shown in the immunoblot as a band at 74 kD. A further band can occasionally be observed at 54 kD, near Jo-1. The further characterisation of mitrochondrial M2-epitopes can be achieved using the liver immunoblot (Catalogue no. IB 150). |
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| ACA
(anti Centromer) ACAs are clinically relevant in cases of CREST syndrome and possibly scleroderma. Comments: Of the two ACA bands (17 kD and 80 kD), often only the band at 17 kD can be detected. It is located very close to the Sm-D band. |
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| Histone Anti-histone (H1) antibodies are shown as a band at 40 kD. Histone antibodies are clinically associated with the following diseases: · medication-induced LE · systemic lupus erythematosus (SLE) · seropositive rheumatoid arthritis · ANA-positive progressive systemic scleroderma Comment: Nonspecific marker of autoimmune diseases. |
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| Jo-1 Jo-1 Antikörper stellen sich als Bande bei 55 kD dar. Der Nachweis von Jo-1 Antikörper ist klinisch ein hochspezifischer Marker für die Dermatomyositis oder Polymyositis. Bemerkungen: Auch schwache Banden sind für die Diagnose relevant. |
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| Ku Anti-Ku antibodies are shown in the immunoblot as a band at 86 kD. A further band may also appear at 76 kD. Comment: The incidence of Ku antibodies is approx.15 % in cases of scleroderma or scleroderma/polymyositis overlap syndrome. |
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| NuMA Anti NuMA antibodies are shown in the immunoblot as a band at 250 kD. NuMA antibodies are only very rarely found. They are observed in patients suffering from Sjögren´s syndrome, rheumatoid arthritis, collagenosis and scleroderma. Comments: None |
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| PmScl PmScl is a complex of 10 nucleolar antigens.One of the most important marker proteins has a molecular weight of 30 kD. PmScl antibodies are a rare but highly specific marker for the scleroderma /polymyositis overlap syndrome. Comments: The band detected in the immunoblot at 30 kD is the principal epitope, but only one of approx. ten PmScl bands. The PmScl bands appear at the same height as the SmB´ bands. The scanner cannot differentiate between them both. Please take into account the following interpretation aid: If two bands appear (e.g. SmB and SmB´or SmB and PmScl) then it is always Sm B, SmB´. If only one band appears, SmB´or PmScl then it is always PmScl. |
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| U-1
RNP (70 kD) Antibodies against U-1 RNP are always shown as a triple band (triplet). If they appear alone (without Sm), antibodies against U-1 RNP are a pathognomonic marker for mixed connective tissue disease (MCTD) with a high clinical sensitivity of 95 % -100 %. Comments: In the interpretation by scanner, problems may arise in identifying the triplet. A clearly visible triplet cannot be completely detected by the scanner, it then only analyses one or two bands. This problem occurs chiefly with a raised background colour. Close to the RNP triplet is a nonspecific band (heat shock proteins). |
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| RNP
a und c (33 kD und 22 kD) Appear as broad bands at 22 kD and 33 kD . An isolated occurrence of these bands points to SLE and indicates an existing or developing lupus nephritis. However, if they appear in combination with the 70 kD RNP triplet, they are an accepted indication of SLE or the mixed connective tissue diseases (MCTD). The appearance of RNP 33 kD and 22 kD as well as Sm are an important indication of existing lupus nephritis (a serious complication of SLE). Comments: A nonspecific band exists close to the 33 kD band. |
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| Rib-P
(rRNP) Rib-P antibodies are shown in the immunoblot as a band at 38 kD. Antibodies can also rarely be found at 19 and 18 kD. These antibodies are symptomatic of a psychotic involvement in SLE. Comments: None |
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| Scl
70 Scl 70 antibodies are shown in the blot as a band (and sometimes as a fine double band) at 100 kD. Scl 70 antibodies are highly specific markers of progressive systemic sclerosis (PSS). These antibodies appear more rarely in patients suffering from circumscribed or linear scleroderma. Comments: Close to the Scl 70 band is another band with a nucleolar immunofluorescent pattern. |
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| Sm Consists of three proteins SmB‘ (29 kD), SmB (28 kD), SmD (19 kD). A double band for the SmBB‘ complex must be visible in the blot at 28 and 29kD for the results to be assessed as positive. Sm is a typical marker for SLE, these antibodies can also occasionally occur in cases of rheumatoid arthritis. Comments: The scanner cannot differentiate between PmScl and Sm B´ (see PmScl). 2 bands at this location are always Sm BB´. |
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| SSA
/ Ro (60kD) SSA appears in the immunoblot as two bands (SSA-60 kD and SSA-52 kD). The isolated occurrence of the SSA-60 kD band points to SLE , whereas in combination with SSA-52 kD it points to SLE or Sjögren´s syndrome. Comment: Close to the 60 kD band lies the HSP 60 (Heat shock protein). |
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| SSA
/ (52kD) Typical of Sjögren´s syndrome. Important marker of neonatal lupus and congenital heartblock. |
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| SSB
/ La (45kD) Anti SSB antibodies are shown in the immunoblot as a band at 45 kD. The SSB band appears in most cases together with the SSA-52 kD. SSB is a typical marker of Sjögren´s syndrome. In combination with SSA 52 kD, SSB is a marker of neonatal lupus and congenital heartblock. |
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| Besides the characterized
antigens, the immunoblot also contains all other Hep2 cell antigens.
Due to the fact that characterized marker sera for these antigens
are not available in the necessary quantity, the antigens cannot be
included in routine quality controls. |
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AID
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