Immunofixation Electrophoresis ( IFE) is a method to detect monoclonal band (M-band). The M-band consist of monoclonal proteins (M-proteins), which are abnormal proteins produce by continous proliferation of plasma cells. The purpose of implementing IFE is to investigate the M-band, that is to know which particular immunoglobulin heavy chains, gamma(IgG), alpha(IgA) and mu(IgM) and light chains (kappa or lambda) that make up the M-proteins. In the laboratory that I am attached to, usually the test that require IFE is myeloma test. In addition, IFE also provides urine protein electrophoresis, which is useful in the study of renal excretion of proteins.
Steps involve:
1. Proteins in the samples (urine and serum) are separated by electrophoresis on agarose gel.
2. Immunofixation of electrophoresed proteins includes, the monospecific antisera diffuse into
agarose gel and bind to the antigens (proteins), if present. Thus, resulting in the precipitation
of the antigens.
3. The unprecipitated antigens are removed from the gel by blotting and washing.
4. The precipitated antigens are stained and can be evaluated visually. M-band is seen when one
of the fractions is dark in colour.
IFE only provides qualitative determination of results.
Siti Shahimah Bte Samat
0702717J
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Hi. Erm sorry to ask you that, i still dont understand what is the purpose of knowing which heavy chain( gamma,alpha and mu) makes up the heavy chain?
ReplyDeleteJennifer
The blotting and washing step, to remove unprecipitated antigens, uses what kind of reagents? Is it the same as western blotting?
ReplyDeleteLiyana
0703827F
Hi Siti,
ReplyDeleteWhat do you do next after detecting M-band?
Rachel :)
hi siti,
ReplyDeletedo you induce the use of any quality control/ladder for the electrophoresis?
jiahui
tg01 group 2 0703605f
hi siti, you mention renal excretion of proteins. Under what conditions will that happen? what is the clinical significance?
ReplyDeletezi shuang
Siti,
ReplyDelete(1)Is this a native electrophoresis or a SDS-electrophoresis?
(2) Are there any difference between Imunofixation and western blot?
Li Yinliang Alex 0704894E
TG02 Group 8
26 October 2009
to jeniffer: it is for classification purposes- e.g. monoclonal gammopathy of IgM indicates Waldenstrom's disease
ReplyDeleteto Liyana: For Immunofixation, it requires no blotting. The blotting that i mentioned above is to remove excess antisera. Sorry for the confusion. It is not the same as western blot.
to Rachel: report results and do quantification.
to jiahui: Yes. the lab do QC for electrophoresis.
to zi shuang: urine electropohoresis is ordered when protein is detected in urine. however,the presence of protein sometimes do not indicate clinical significance. Kidney damage/ disease( e.g. diabetes, Bences Jones protein) may cause protein to be present in the urine.
to alex: 1. i am sure that it is not SDS electrophoresis. in this lab that i am attached to, we use hydragel 7 kit ( sebia). we use the ready-made agarose gel which consist of agarose, tris-barbital buffer and other additives too.
2. the difference is that immunofixation involves putting antisera directly onto the electrophoresed proteins while western blot involves the use of a membrane which is labeled with antibodies.