Serum protein electrophoresis (SPEP or SPE) separates the complex mixture of serum proteins into distinct fractions based on their electrophoretic mobility. It is a primary screening tool for multiple myeloma and other plasma cell dyscrasias.

Principle

Serum proteins are negatively charged at alkaline pH (pH 8.6) and migrate toward the anode when an electric field is applied. The rate of migration depends on the protein’s net charge, size, and shape, as well as the supporting medium (agarose gel or cellulose acetate).

On agarose gel at pH 8.6, serum proteins separate into five major fractions (from anode to cathode):

1. Albumin (the largest peak, ~55–65% of total protein), migrates fastest.
2. α1-globulins (α1-antitrypsin, α1-acid glycoprotein, ~2–4%), the smallest fraction.
3. α2-globulins (haptoglobin, α2-macroglobulin, ceruloplasmin, ~7–10%).
4. β-globulins (transferrin, complement C3, β-lipoprotein, ~8–13%), may split into β1 and β2 bands.
5. γ-globulins (immunoglobulins IgA, IgD, IgE, IgG, IgM, ~10–20%), the broad, diffuse band at the cathodal end.

Procedure

1. Apply 5–10 µL of serum to the application point on an agarose gel.
2. Electrophorese at 100–200 V for 20–40 minutes (until the albumin front has migrated 4–6 cm).
3. Fix the gel in acid-alcohol solution to precipitate proteins.
4. Stain with Amido Black, Ponceau S, or Coomassie Blue.
5. Destain and dry the gel.
6. Scan with a densitometer to produce a quantitative densitometric tracing.
7. Calculate the percentage of each fraction by integrating the area under the curve.

Total protein is measured separately by biuret assay. The absolute concentration of each fraction is calculated as: percentage × total protein.

Interpretation

Normal pattern: a tall, narrow albumin peak; small α1 peak; slightly larger α2 peak; β peak(s) of variable height; a broad, polyclonal γ-globulin peak.

Monoclonal gammopathy: a tall, narrow, spike-like band (M-spike, monoclonal protein) appears most commonly in the γ region, sometimes in β or α2. The M-spike represents a monoclonal immunoglobulin (usually IgG or IgA) produced by a single clone of plasma cells. It is the hallmark of multiple myeloma, Waldenström macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), and AL amyloidosis.

Polyclonal hypergammaglobulinemia: a broad, diffuse increase in γ-globulins, seen in chronic inflammation, autoimmune diseases (SLE, rheumatoid arthritis), chronic liver disease, and HIV infection.

Hypogammaglobulinemia: decreased γ-globulins, seen in primary immunodeficiencies (CVID, X-linked agammaglobulinemia), secondary immunodeficiency, or nephrotic syndrome.

Acute phase response: decreased albumin, increased α1 and α2 globulins (α1-antitrypsin, haptoglobin). The α1 fraction is a sensitive early marker of inflammation.

Nephrotic syndrome: decreased albumin, increased α2 and β globulins (due to loss of small proteins in urine and compensatory synthesis of larger proteins).

Immunofixation

If an M-spike is detected or suspected, immunofixation electrophoresis (IFE) is performed to identify the immunoglobulin heavy chain (IgG, IgA, IgM, IgD, IgE) and light chain (κ, λ) type. IFE uses specific antisera applied to a replicate gel after electrophoresis:

Patient serum is electrophoresed in six lanes on a gel. After electrophoresis, a specific antiserum is applied to each lane (anti-γ, anti-α, anti-μ, anti-κ, anti-λ, and a fixative control). The gel is washed to remove unprecipitated proteins, stained, and inspected. A monoclonal immunoglobulin appears as a discrete band in one heavy-chain lane and one light-chain lane, confirming both the identity and the clonal restriction of the light chain.

Urine Protein Electrophoresis

Bence Jones proteins (free monoclonal light chains) are detected by urine protein electrophoresis. Because the light chain concentration in urine may be low, 50–100× concentration of the urine sample is typically required. Immunofixation of the urine is performed with anti-κ and anti-λ antisera. The presence of a monoclonal free light chain in urine is diagnostic for light-chain multiple myeloma or AL amyloidosis.