Detection of oligoclonal IgM in cerebrospinal fluid
Authors:
D. Zeman 1,2; P. Kušnierová 1,2
Authors‘ workplace:
Katedra biomedicínských oborů, Lékařská fakulta, Ostravská univerzita
1; Oddělení klinické biochemie, Ústav laboratorní diagnostiky, Fakultní nemocnice Ostrava
2
Published in:
Klin. Biochem. Metab., 24, 2016, No. 3, p. 141-146
Overview
Objective:
Improvement of the procedure used for cerebrospinal fluid (CSF) and serum oligoclonal IgM detection.
Design:
Methodical study
Material and Methods:
Isoelectric focusing (IEF) in agarose gel followed by affinity immunoblotting. After choosing the best variant from the protocols tested, 35 paired CSF and serum samples were examined.
Results:
Improvement of IgM separation has been achieved by using narrower pH gradient (4-8), prefocusing step and cathodic application of samples. Efforts to separate IgM in native CSF or serum samples have been unsuccessful. Satisfactory results have been obtained using sample pretreatment with dithiothreitol, optionally followed by acrylamide pretreatment. Intrathecal synthesis of oligoclonal IgM was demonstrated in 11 out of 35 patients. Samples of symptomatic controls (n=3) or patients with a proved diagnosis of non-inflammatory neurological disease (n=9) were all negative. Oligoclonal IgM result correlated significantly with intrathecal IgM synthesis estimated either by Reiber´s (P=0.007) or Auer´s formula (P=0.026).
Conclusion:
The method described in this article enables sensitive and reproducible detection of oligoclonal IgM bands separated by IEF. Further research should address the optimisation of sample pretreatment as well as experiments aimed at separation of IgM in untreated cerebrospinal fluid and serum samples.
Keywords:
oligoclonal IgM, isoelectric focusing, affinity immunoblotting, calculation of intrathecal synthesis.
Sources
1. Kaiser, R. Affinity immunoblotting: rapid and sensitive detection of oligoclonal IgG, IgA and IgM in unconcentrated CSF by agarose isoelectric focusing. J. Neurol. Sci. 1991, 103, p. 216-225.
2. Sindic, C. J., Monteyne, P., Laterre, E. C. Occurrence of oligoclonal IgM bands in the cerebrospinal fluid of neurological patients: an immunoaffinity-mediated capillary blot study. J. Neurol. Sci. 1994, 124, p. 215-219.
3. Rijcken, C. A., Thompson, E. J., Teelken, A. W. An improved, ultrasensitive method for the detection of IgM oligoclonal bands in cerebrospinal fluid. J. Immunol. Methods 1997, 203, p. 167-169.
4. Villar, L. M., Masjuán, J., González-Porqué, P. et al. Intrathecal IgM synthesis is a prognostic factor in multiple sclerosis. Ann. Neurol. 2003, 53, p. 222-226.
5. Villar, L. M., González-Porqué, P., Masjuán, J., Alvarez-Cermeño, J. C., Bootello, A., Keir, G. A sensitive and reproducible method for the detection of oligoclonal IgM bands. J. Immunol. Methods 2001, 258, p. 151-155.
6. Villar, L. M., Sádaba, M. C., Roldán, E. et al. Intrathecal synthesis of oligoclonal IgM against myelin lipids predicts an aggressive disease course in MS. J. Clin. Invest. 2005, 115, p. 187-194.
7. Sobek, O., Adam, P., Koudelková, M., Štourač, P., Mareš, J. Algoritmus vyšetření likvoru v návaznosti na doporučení Sekce neuroimunologie a likvorologie České neurologické společnosti JEP. Cesk. Slov. Neurol. N. 2012, 75/108 (2), p. 159-163.
8. Deisenhammer, F., Bartoš, A., Egg, R. et al. Routine cerebrospinal fluid (CSF) analysis. In: Gilhus, N.E., Basrnes, M.P., Brainin, M. (Eds.) European Handbook of Neurological Management: Volume 1, 2nd Edition. Blackwell Publishing Ltd. 2011, p. 5-17.
9. Wick, M. (Ed.). Ausgewählte Methoden der Liquordiagnostik und Klinischen Neurochemie, 3. vydání, 2014, dostupné na: www.dgln.de (sekce Empfehlungen – Methodenkatalog)
10. Kelbich, P., Vachová, M., Kotalová, J., Vondráčková, S., Čujková, O. Detekce intratékální syntézy imunoglobulinů – vlastní zkušenosti. Cesk. Slov. Neurol. N. 2014, 77/110 (Suppl), p. S44.
11. Richard, S., Miossec, V., Moreau, J. F., Taufin, J. L. Detection of oligoclonal immunoglobulins in cerebrospinal fluid by an immunofixation-peroxidase method. Clin. Chem. 2002, 48, p. 165-173.
12. Sindic, C. J., Laterre, E. C. Oligoclonal free kappa and lambda bands in the cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases. An immunoaffinity-mediated capillary blot study. J Neuroimmunol 1991, 33, p. 63-72.
13. Zeman, D., Vaníčková, Z., Benáková, H., Havrdová, E. Volné lehké řetězce typu kappa v likvoru a séru. Klin. Biochem. Metab. 2002, 10 (31), p. 98-102.
14. Nováčková, L., Zeman, D. Detection of oligoclonal IgG bands in cerebrospinal fluid and serum: comparison between commercial immunofixation method and home-made affinity immunoblotting method and evaluation of interobserver agreement. Klin. Biochem. Metab. 2011, 19 (40), p. 229-233.
15. Zeman, D., Hradílek, P., Kušnierová, P. et al. Oligoclonal free light chains in cerebrospinal fluid as markers of intrathecal inflammation. Comparison with oligoclonal IgG. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc, Czech Repub. 2015, 159, p. 104-114.
16. Bojková, J. Detekce nízkých koncentrací paraproteinů v séru a moči metodou izoelektrické fokusace s afinitním imunoblottingem. Diplomová práce. Ústav zdravotnic-kých disciplín, Vysoká škola zdravotníctva a sociálnej práce sv. Alžbety, Bratislava, 2015, p. 1-77.
17. Reiber, H. Flow rate of cerebrospinal fluid (CSF) – a concept common to normal blood-CSF barrier function and to dysfunction in neurological diseases. J. Neurol. Sci. 1994, 122, p. 189-203.
18. Auer, M., Hegen, H., Zeileis, A., Deisenhammer, F. Quantitation of intrathecal immunoglobulin synthesis – a new empirical formula. Eur. J. Neurol. 2016, p. 713-721.
19. Westermeier, R. Agarose IEF. In: Westermeier R. Electrophoresis in practice. Fourth, revised and enlarged edition. Willey-VCH, Weinheim 2005: p. 189-196.
20. Cornell, F. N. Isoelectric focusing, blotting and probing methods for detection and identification of monoclonal proteins. Clin. Biochem. Rev., 2009, 30, p. 123-130.
21. Rosén, A., Ek, K., Åman, P. Agarose isoelectric focusing of native human immunoglobulin M and α2-macroglobulin. J. Immunol. Methods 1979, 28, p. 1-11.
22. Jackson, D. E., Skandera, C. A., Owen, J., Lally, E. T., Montgomery, P. C. Isoelectric focusing of IgA and IgM in composite acrylamide-agarose gels. J. Immunol. Methods 1980, 36, p. 315-324.
23. Chiodi, F., Sidén, Å., Ösby, E. Isoelectric focusing of monoclonal immunoglobulin G, A and M followed by detection with the avidin-biotin system. Electrophoresis 1985, 6, p. 124-128.
24. Schibeci, A., Wade, A. W., Depew, W. T., Szewczuk, M. R. Analysis of serum antibody repertoires by isoelectric focusing and capillary blotting onto nitrocellulose paper. J. Immunol. Methods 1986, 89, p. 201-205.
25. Herbert, B. Some practices and pitfalls of sample pre-paration for isoelectric focusing in proteomics. In: Garfin D, Ahuja S (Eds.). Handbook of Isoelectric Focusing. Elsevier, Amsterdam 2005, p. 147-164.
26. Wang, Z. Y., Hansen, K., Sidén, Å., Cruz, M. Intrathecal synthesis of anti-Borrelia burgdorferi antibodies in neuroborreliosis: a study with special emphasis on oligoclonal IgM antibody bands. Scand. J. Immunol. 1993, 37, p. 369-376.
27. Zeman, D., Kušnierová, P., Gottwaldová, J. et al. Úskalí kvantifikace IgM v likvoru. Klin. Biochem. Metab. 2012, 20 (41), p. 136-144.
28. Kelbich, P. Jsem skeptický vůči výpočtům intrathe-kální syntézy imunoglobulinů. (Editorial). Klin. Biochem. Metab. 2012, 20 (41), p. 133.
29. Keren, D. F., Schroeder, L. Challenges of measuring monoclonal proteins in serum. Clin. Chem. Lab. Med. 2016, Feb 24. doi: 10.1515/cclm-2015-0862. [Epub ahead of print]
30. Schneider, R., Euler, B., Rauer, S. Intrathecal IgM-synthesis does not correlate with the risk of relapse in patients with a primary demyelinating event. Eur. J. Neurol. 2007, 14, p. 907-911.
Labels
Clinical biochemistry Nuclear medicine Nutritive therapistArticle was published in
Clinical Biochemistry and Metabolism
2016 Issue 3
Most read in this issue
- Detection of oligoclonal IgM in cerebrospinal fluid
- Vitamins in critically ill patients.
- Advances in immunoassays by luminescence and electrochemical detection
- New diagnostic criteria of multiple myeloma