Reviewer's report
Title:Analysis of the clinical relevance of antimitochondrial antibodies to the
beta- and gamma-subunits of the F1F0-ATPase in patients with primary biliary
cirrhosis
Version: 1Date:5 July 2012
Reviewer: Lars Komorowski
Reviewer's report:
The authors describe the prevalence an clinical relevance of autoantibodies
against subunits of mitchondrial F1F0-ATPase in primary biliary cirrhosis. For this
purpose, they employ two ELISA systems based on proteins expressed in E. coli
and purified under denaturing conditions. They proclaim an additional value for
the initial serological diagnosis of the disease but neglect the usefulness of the
autoantibodies for activity monitoring or prognosis of disease outcome.
1) Anti-M2 determination
The authors describe the use of a preparation of the 2-oxoacid dehydrogenase
complex (ODC) from beef heart mitochondria but its use – probably as an
antigen in an immunoassay for the determination of anti-M2 – and the actual
immunoassay that was conducted for the determination of anti-M2 are obscure.
Moreover, the classical protocol for the preparation of ODC mainly enriches the
pyruvate dehydrogenase complex (PDC) but the proportions of the
branched-chain 2-oxo-acid dehydrogenase complex (BCOADC) and the
2-oxo-glutarate dehydrogenase complex (OGDC) vary. Taken together with the
importance of the E2 subunits of BCOADC and OGDC for a competent
determination of anti-M2 – as has been shown in several studies – this results in
a reference assay that can not be considered state-of-the-art. This probaly
explains the lower clinical sensitivity of this assay compared to anti-mitochondrial
autoantibodies (AMA) determined by indirect immunofluorescence. The authors
should employ either the three E2 subunits seperately or the fusion protein
consisting of them (MIT3) as the basis of the reference assay. Otherwise,
samples must not be deemed anti-M2 negative. Therefore, the statement that the
determination of anti-beta- and/or gamma-antibodies increases the sensitivity of
the serological diagnosis is not valid.
2) Relation of immunofluorescence pattern and positivity in the ELISA
In the introduction part, the authors state that a considerable proportion of sera
generating the anti-mitochondrial autoantibody (AMA) pattern in indirect
immunofluoresce are negative for anti-M2 in their reference assay (s. #1). They
indicate that in these cases the beta- and gamma-subunits of the F1F0-ATPase
– located in mitochondrial membrane – are targeted, resulting in the AMA
staining pattern. However, in their experiments AMA negative sera are also
positive but the interpretation is never corrected.
3) Data of PBC patients vs. healthy controls
In Figure 1 the authors show the consolidated data of their ELISA experiments
employing sera from 59 PBC patients and 41 healthy controls. In order to let the
reader get a better impression the indvidual data of each samples should be
given in a dot plot. Although the given comparison of the mean values
demonstrates statistical signifcant differences the data – even in this
consolidated format – indicate a highly impractical narrow margin between the
two groups. Moreover, disease controls with high probabilty of “false” reactivities
are missing which obscures the true value of the autantibodies for serological
diagnosis.
In conclusion the manuscript can not be considered fit for publication.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Reviewer's report
Title:Analysis of the clinical relevance of antimitochondrial antibodies to the
beta- and gamma-subunits of the F1F0-ATPase in patients with primary biliary
cirrhosis
Version: 1Date:5 July 2012
Reviewer: Lars Komorowski
Reviewer's report:
The authors describe the prevalence an clinical relevance of autoantibodies
against subunits of mitchondrial F1F0-ATPase in primary biliary cirrhosis. For this
purpose, they employ two ELISA systems based on proteins expressed in E. coli
and purified under denaturing conditions. They proclaim an additional value for
the initial serological diagnosis of the disease but neglect the usefulness of the
autoantibodies for activity monitoring or prognosis of disease outcome.
1) Anti-M2 determination
The authors describe the use of a preparation of the 2-oxoacid dehydrogenase
complex (ODC) from beef heart mitochondria but its use – probably as an
antigen in an immunoassay for the determination of anti-M2 – and the actual
immunoassay that was conducted for the determination of anti-M2 are obscure.
Moreover, the classical protocol for the preparation of ODC mainly enriches the
pyruvate dehydrogenase complex (PDC) but the proportions of the
branched-chain 2-oxo-acid dehydrogenase complex (BCOADC) and the
2-oxo-glutarate dehydrogenase complex (OGDC) vary. Taken together with the
importance of the E2 subunits of BCOADC and OGDC for a competent
determination of anti-M2 – as has been shown in several studies – this results in
a reference assay that can not be considered state-of-the-art. This probaly
explains the lower clinical sensitivity of this assay compared to anti-mitochondrial
autoantibodies (AMA) determined by indirect immunofluorescence. The authors
should employ either the three E2 subunits seperately or the fusion protein
consisting of them (MIT3) as the basis of the reference assay. Otherwise,
samples must not be deemed anti-M2 negative. Therefore, the statement that the
determination of anti-beta- and/or gamma-antibodies increases the sensitivity of
the serological diagnosis is not valid.
2) Relation of immunofluorescence pattern and positivity in the ELISA
In the introduction part, the authors state that a considerable proportion of sera
generating the anti-mitochondrial autoantibody (AMA) pattern in indirect
immunofluoresce are negative for anti-M2 in their reference assay (s. #1). They
indicate that in these cases the beta- and gamma-subunits of the F1F0-ATPase
– located in mitochondrial membrane – are targeted, resulting in the AMA
staining pattern. However, in their experiments AMA negative sera are also
positive but the interpretation is never corrected.
3) Data of PBC patients vs. healthy controls
In Figure 1 the authors show the consolidated data of their ELISA experiments
employing sera from 59 PBC patients and 41 healthy controls. In order to let the
reader get a better impression the indvidual data of each samples should be
given in a dot plot. Although the given comparison of the mean values
demonstrates statistical signifcant differences the data – even in this
consolidated format – indicate a highly impractical narrow margin between the
two groups. Moreover, disease controls with high probabilty of “false” reactivities
are missing which obscures the true value of the autantibodies for serological
diagnosis.
In conclusion the manuscript can not be considered fit for publication.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Please review this paper.
user
Reviewer's report
Title:Analysis of the clinical relevance of antimitochondrial antibodies to the
beta- and gamma-subunits of the F1F0-ATPase in patients with primary biliary
cirrhosis
Version: 1Date:5 July 2012
Reviewer: Lars Komorowski
Reviewer's report:
The authors describe the prevalence an clinical relevance of autoantibodies
against subunits of mitchondrial F1F0-ATPase in primary biliary cirrhosis. For this
purpose, they employ two ELISA systems based on proteins expressed in E. coli
and purified under denaturing conditions. They proclaim an additional value for
the initial serological diagnosis of the disease but neglect the usefulness of the
autoantibodies for activity monitoring or prognosis of disease outcome.
1) Anti-M2 determination
The authors describe the use of a preparation of the 2-oxoacid dehydrogenase
complex (ODC) from beef heart mitochondria but its use – probably as an
antigen in an immunoassay for the determination of anti-M2 – and the actual
immunoassay that was conducted for the determination of anti-M2 are obscure.
Moreover, the classical protocol for the preparation of ODC mainly enriches the
pyruvate dehydrogenase complex (PDC) but the proportions of the
branched-chain 2-oxo-acid dehydrogenase complex (BCOADC) and the
2-oxo-glutarate dehydrogenase complex (OGDC) vary. Taken together with the
importance of the E2 subunits of BCOADC and OGDC for a competent
determination of anti-M2 – as has been shown in several studies – this results in
a reference assay that can not be considered state-of-the-art. This probaly
explains the lower clinical sensitivity of this assay compared to anti-mitochondrial
autoantibodies (AMA) determined by indirect immunofluorescence. The authors
should employ either the three E2 subunits seperately or the fusion protein
consisting of them (MIT3) as the basis of the reference assay. Otherwise,
samples must not be deemed anti-M2 negative. Therefore, the statement that the
determination of anti-beta- and/or gamma-antibodies increases the sensitivity of
the serological diagnosis is not valid.
2) Relation of immunofluorescence pattern and positivity in the ELISA
In the introduction part, the authors state that a considerable proportion of sera
generating the anti-mitochondrial autoantibody (AMA) pattern in indirect
immunofluoresce are negative for anti-M2 in their reference assay (s. #1). They
indicate that in these cases the beta- and gamma-subunits of the F1F0-ATPase
– located in mitochondrial membrane – are targeted, resulting in the AMA
staining pattern. However, in their experiments AMA negative sera are also
positive but the interpretation is never corrected.
3) Data of PBC patients vs. healthy controls
In Figure 1 the authors show the consolidated data of their ELISA experiments
employing sera from 59 PBC patients and 41 healthy controls. In order to let the
reader get a better impression the indvidual data of each samples should be
given in a dot plot. Although the given comparison of the mean values
demonstrates statistical signifcant differences the data – even in this
consolidated format – indicate a highly impractical narrow margin between the
two groups. Moreover, disease controls with high probabilty of “false” reactivities
are missing which obscures the true value of the autantibodies for serological
diagnosis.
In conclusion the manuscript can not be considered fit for publication.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
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