Autoimmunity, 1992, Vol. 13, pp. 173-174
0 1992 Hanvood Academic Publishers GmbH
Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Correspondence
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
FAILURE TO DETECT AUTOANTIBODIES SPECIFIC FOR IDDM BY WESTERN BLOTTING Sir, The problems associated with the reproducibility of the ICA immunofluorescence technique’ have caused many research groups to attempt to extract the cytoplasmic antigen from pancreatic islet cells and use alternative techniques to determine ICAs. One such technique involved immunoblot analysis after separation of RIN m5F cell proteins using one- or twodimensional SDS-polyacrylamide gel electrophoresis2. Sera from newly diagnosed diabetic patients reacted with multiple islet antigens at high titres, whilst normal control sera bound a single antigen at a much weaker dilution. We have not been able to reproduce these results, finding poor discrimination between sera from
newly diagnosed diabetics and normal controls. We compared a Sigma anti-human IgG conjugate with the purified anti-human IgG preparation from Kirkegaard and Perry recommended by Karounos et al.’ but found the latter much weaker; when both conjugates were used at equivalent concentrations, the same bands were detected. Karounos et al. appear to have assayed the sera from IDDM patients and controls on separate immunoblots. It can be difficult to be certain of differences between the sera unless assayed on the same blot. The figure shows a single immunoblot on which we compared sera from normal control and newly diagnosed diabetic patients using both the Kirkegaard and Perry and the Sigma conjugates at equivalent concentrations. The
K Da 4116
4
49
4 36
1 2 3 4 5 6 7 8 9 1011 12 13 141516 Figure 1 Immunoblot of diabetic and normal sera diluted 1:lOO for their ability to bind RIN m5F antigens. Lanes 1-8 normal sera, lines 9-16 newly diagnosed diabetic sera. Odd numbered lanes were incubated with the Sigma conjugate, even numbered lanes were incubated with the Kirkegaard and Perry conjugate, Molecular weight markers are indicated in kilodaltons on the right.
173
174
CORRESPONDENCE
Kirkegaard and Perry conjugate was less sensitive and all sera reacted similarly with the R I N m5F cell extract . In conclusion, sera from newly diagnosed IDDM patients and normal controls have been evaluated with no apparent difference in immunoreactivity.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
A. B. TUCK, A. M. PETLEY and T. J. WILKIN, Endocrine Section, Medicine 11, Southampton General Hospital, Southampton SO9 4 X Y .
Refeiences I . Bonifacio E, Boitard C, Gleichmann H , Shattock M, Molcnaar J, Bottazzo GF and co-authors. Assessment of precision, concordance. specificity and sensitivity of islet cell antibody measurement in 4 I assays. Diuhrrologiu 1990; 33: 73 1-736 2. Karounos D, Nell L. Thomas J. Autoantibodies present at onset of type I diabetes recognise multiple islet cell antigens. Autoimmunity 1990; 2: 79-9 1
0 1992 Hanvood Academic Publishers GmbH
Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Correspondence
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
FAILURE TO DETECT AUTOANTIBODIES SPECIFIC FOR IDDM BY WESTERN BLOTTING Sir, The problems associated with the reproducibility of the ICA immunofluorescence technique’ have caused many research groups to attempt to extract the cytoplasmic antigen from pancreatic islet cells and use alternative techniques to determine ICAs. One such technique involved immunoblot analysis after separation of RIN m5F cell proteins using one- or twodimensional SDS-polyacrylamide gel electrophoresis2. Sera from newly diagnosed diabetic patients reacted with multiple islet antigens at high titres, whilst normal control sera bound a single antigen at a much weaker dilution. We have not been able to reproduce these results, finding poor discrimination between sera from
newly diagnosed diabetics and normal controls. We compared a Sigma anti-human IgG conjugate with the purified anti-human IgG preparation from Kirkegaard and Perry recommended by Karounos et al.’ but found the latter much weaker; when both conjugates were used at equivalent concentrations, the same bands were detected. Karounos et al. appear to have assayed the sera from IDDM patients and controls on separate immunoblots. It can be difficult to be certain of differences between the sera unless assayed on the same blot. The figure shows a single immunoblot on which we compared sera from normal control and newly diagnosed diabetic patients using both the Kirkegaard and Perry and the Sigma conjugates at equivalent concentrations. The
K Da 4116
4
49
4 36
1 2 3 4 5 6 7 8 9 1011 12 13 141516 Figure 1 Immunoblot of diabetic and normal sera diluted 1:lOO for their ability to bind RIN m5F antigens. Lanes 1-8 normal sera, lines 9-16 newly diagnosed diabetic sera. Odd numbered lanes were incubated with the Sigma conjugate, even numbered lanes were incubated with the Kirkegaard and Perry conjugate, Molecular weight markers are indicated in kilodaltons on the right.
173
174
CORRESPONDENCE
Kirkegaard and Perry conjugate was less sensitive and all sera reacted similarly with the R I N m5F cell extract . In conclusion, sera from newly diagnosed IDDM patients and normal controls have been evaluated with no apparent difference in immunoreactivity.
Autoimmunity Downloaded from informahealthcare.com by McMaster University on 10/29/14 For personal use only.
A. B. TUCK, A. M. PETLEY and T. J. WILKIN, Endocrine Section, Medicine 11, Southampton General Hospital, Southampton SO9 4 X Y .
Refeiences I . Bonifacio E, Boitard C, Gleichmann H , Shattock M, Molcnaar J, Bottazzo GF and co-authors. Assessment of precision, concordance. specificity and sensitivity of islet cell antibody measurement in 4 I assays. Diuhrrologiu 1990; 33: 73 1-736 2. Karounos D, Nell L. Thomas J. Autoantibodies present at onset of type I diabetes recognise multiple islet cell antigens. Autoimmunity 1990; 2: 79-9 1
