Monoclonal Immunoglobulin (Ig), Monoclonal antibody, Immunofixation Electrophoresis (IFE)

Sample

1. IFE (Immunofixation electrophoresis) can be done on the serum of the patient.
2. Other fluids are CSF, urine and biological fluids.
3. 24 hours of urine is collected according to the instructions.
4. Can store the sample at 2 to 8 °C for 5 days.

Precautions

1. The fresh serum is the choice of sample.
2. The fasting sample is preferred.
3. Avoid anticoagulant use.
4. Centrifuge the sample immediately for 15 minutes.

Purpose of the test

1. To identify the monoclonal gammopathy.
2. To monitor the treatment with monoclonal gammopathy.
3. To find the light chains / heavy chain of immunoglobulin in the urine.
4. Monoclonal gammopathy of undetermined significance (MGUS)
5. Waldenstrom’s macroglobulinemia.
6. Multiple Myeloma (used more than 100 years).
7. Amyloidosis.

Pathophysiology

1. Immunofixation electrophoresis is the study of protein antigens, and their split products and evaluation of myeloma.
   1. This was described in 1964.
   2. Immunofixation electrophoresis is replacing the electrophoresis because of its rapidity and ease of interpretation.
   3. The principle is the same in both processes.
2. Monoclonal immunoglobulin (Ig) consists of heavy and light chains.
   1. These are purified antibodies cloned from a single cell.
   2. These antibodies will binds to surface antigen.
   3. The production of the monoclonal antibodies in the animals takes 3 to 6 months.

3. IFE identify the monoclonal Ig and also identify light chain or heavy chains.

4. By electrophoresis, there is a spike of monoclonal Ig (Antibody).

5. Waldenstrom’s macroglobulinemia also shows a monoclonal spike.
   1. Above both diseases give light chains excretion in the urine.
   2. IFE can also find that the spike is due to a light chain or heavy chain.
6. Chronic diseases give rise to polyclonal bands of these immunoglobulins.
7. Non-malignant monoclonal immunoglobulin seen in:
   1. 5% in people over the age of 75 years of age.
   2. These are lower in the concentration than the malignant bands.

Procedure and Principle of Immunofixation electrophoresis (IFE)

1. This IFE is a two-stage procedure:
   1. Agarose gel protein electrophoresis (IEF)
   2. Immunoprecipitation.
2. The sample of the patient is placed in six separate wells on the agarose gel and their major protein group is separated by electrophoresis.
3. One of this track is treated with a chemical fixation solution to fix all the proteins in agarose and create an electrophoresis reference pattern for the specimen.
4. The other five tracks are treated with heavy and light chains antisera which react with individual immunoglobulins in the specimen causing them to Immuno- fix in the agarose.
5. All unreacted proteins are washed from those five tracks out of the gel.
6. Now all 6 tracks are stained to visualized the fixed proteins bands.
   

   Immunofixation Electrophoresis

Normal

• No monoclonal band is identified.

The monoclonal band is seen in:

1. Multiple myelomas show in 99 % of the patients in serum and urine.
2. Waldenstrom macroglobulinemia always shows a serum IgM type monoclonal band.
3. Monoclonal light chains also called κ or Bence Jones protein, seen in the urine of Multiple myeloma cases in 75 % of the patients.
4. Waldenstrom macroglobulinemia also shows light chains in the urine in 75 % of the patients.
5. Amyloidosis can see the light chain or heavy chain in the urine.

Multiple Myeloma signs and symptoms are:

1. Weakness and fatigue.
2. Weakness in the legs.
3. Nausea and vomiting.
4. Recurrent infections.
5. Bone pain in the back or ribs.
6. H/O fracture

Polyclonal bands of Ig are seen in:

1. Chronic infections.
2. Autoimmune diseases.
3. Chronic liver diseases.
4. Amyloidosis.

Monoclonal Ig is seen in urine:

1. Multiple myelomas.
2. Waldenstrom macroglobulinemia.

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