Plasma Proteins and Electrophoresis

An amino acid contains an amino group, a carboxyl group, a hydrogen atom, and a variable side chain attached to an alpha carbon. The side chain determines charge, polarity, and many of the chemical interactions that shape a protein. At physiologic pH, most free amino acids contain both a positively charged amino group and a negatively charged carboxyl group. This dipolar form is a zwitterion. The pH at which a molecule has no net electrical charge is its isoelectric point, or pI.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

A peptide bond joins the carboxyl group of one amino acid to the amino group of another and releases water. Two joined residues form a dipeptide; longer chains form polypeptides. Protein digestion reverses these bonds by hydrolysis. Gastric acid unfolds dietary proteins, and pepsin begins proteolysis. Secretin promotes pancreatic bicarbonate secretion to neutralize acid in the duodenum, and cholecystokinin promotes secretion of pancreatic proteases. Trypsin, chymotrypsin, elastase, and carboxypeptidases produce smaller peptides. Brush-border and intracellular peptidases complete digestion, and transporters carry amino acids and small peptides into enterocytes for delivery to the portal circulation.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Nutritional requirements vary with age, growth, illness, and metabolic defects, so the adult categories below are only a starting point.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Protein structure is described at four levels.

Denaturation changes secondary, tertiary, or quaternary structure and usually impairs function. Peptide-bond cleavage changes primary structure and is proteolysis or degradation. This distinction matters because a denatured protein can retain its amino acid sequence while losing activity or solubility.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Ribosomes translate messenger RNA into polypeptides. Folding, cleavage, glycosylation, phosphorylation, and other post-translational changes produce the mature protein. Hepatocytes make albumin, transferrin, fibrinogen, and many complement and coagulation proteins. Plasma cells make immunoglobulins, and tissue-specific proteins enter blood from other organs.

Proteins can be classified by shape or composition. Globular proteins include albumin, enzymes, and immunoglobulins. Fibrous proteins include collagen and fibrin. A simple protein yields amino acids when completely hydrolyzed. A conjugated protein also contains a nonprotein component. Hemoglobin is a hemoprotein, lipoproteins carry lipid, metalloproteins bind a metal, glycoproteins and proteoglycans contain carbohydrate, and nucleoproteins contain nucleic acid. Immunoglobulins are glycoproteins.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Protein solubility often reaches a minimum near the pI because reduced net charge decreases electrostatic repulsion among molecules. In isoelectric focusing, a protein migrates through a stable pH gradient until it reaches the pH that matches its pI. The method's high resolution allows it to resolve cerebrospinal fluid oligoclonal bands.

Protein turnover continually releases amino acids for reuse or catabolism. Nitrogen balance is the difference between nitrogen intake and nitrogen loss. Transamination transfers an amino group to an alpha-keto acid, and oxidative deamination can release ammonia. The liver converts much of that nitrogen to urea. The remaining carbon skeleton is glucogenic, ketogenic, or both according to the pathway it enters.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

At the alkaline pH commonly used for serum protein electrophoresis, most major serum proteins have a net negative charge. They migrate toward the anode at rates determined mainly by charge, with size, shape, support medium, and electro-osmotic effects also contributing. The familiar fractions are prealbumin, albumin, alpha-1, alpha-2, beta, and gamma.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Measurement methods

Separation and measurement

Agarose gel electrophoresis commonly uses an alkaline buffer near pH 8.4 to 8.8 and separates serum proteins into bands, which are stained and scanned by a densitometer. Capillary zone electrophoresis separates proteins in narrow capillaries and measures absorbance directly as they pass a detector. Both systems commonly report albumin, alpha-1, alpha-2, beta, and gamma fractions, and their fraction boundaries, resolution, and interference patterns differ. High-resolution agarose and capillary methods can resolve additional bands. The number of bands is platform-dependent. Isoelectric focusing separates proteins by pI and is especially useful for restricted immunoglobulin bands in cerebrospinal fluid. Laboratories validate the method, review flagged traces, and establish their own reference intervals.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

An absolute fraction concentration can be calculated from its densitometric percentage and the total protein measured on the same specimen. Suppose albumin is 60% of a total protein concentration of 6.8 g/dL:

albumin fraction = 0.60 × 6.8 g/dL = 4.08 g/dL

This estimate inherits error from the total-protein assay, trace integration, and peak-boundary selection.

Electrophoretic mobility relates migration distance to time and electric field strength:

mobility (cm²/[V·s]) = migration distance (cm) ÷ [time (s) × field strength (V/cm)]

Suppose a band travels 4.2 cm in 4,200 seconds at 10 V/cm:

mobility = 4.2 ÷ (4,200 × 10) = 0.00010 cm²/(V·s)

The calculation describes movement under the stated conditions. It does not make results from different buffers, temperatures, support media, or instruments interchangeable.

Pattern recognition

Electrophoretic patterns narrow the differential. Interpret them with the specimen, the quantitative results, and the clinical context.^{1,71 Bishop ML, et al. Clinical Chemistry. 2023.7 Keren DF, et al. Laboratory detection and initial diagnosis of monoclonal… 2022.}

An inherited or acquired bisalbuminemia can split the albumin peak. The paired peaks have little clinical effect by themselves. Recognizing the pattern keeps an albumin variant from being mistaken for a restricted globulin.^{11 Bishop ML, et al. Clinical Chemistry. 2023.}

Several artifacts can mimic an abnormal pattern. Plasma fibrinogen may form a restricted band near the beta-gamma interface. Hemolysis can add hemoglobin-related bands and complicate haptoglobin interpretation. High C-reactive protein can form a restricted band on some systems. Radiographic contrast, certain antimicrobials, and therapeutic monoclonal antibodies can also alter a trace. Previous results, specimen type, medication history, and confirmatory testing help resolve these findings.^{1,71 Bishop ML, et al. Clinical Chemistry. 2023.7 Keren DF, et al. Laboratory detection and initial diagnosis of monoclonal… 2022.}

A monoclonal protein is an immunoglobulin or immunoglobulin component produced by one clone. The laboratory finding can accompany monoclonal gammopathy of undetermined significance, plasma-cell neoplasms, lymphoplasmacytic disorders, amyloid light-chain amyloidosis, and other conditions. A monoclonal IgM result occurs in several of these disorders, so isotype alone does not establish the diagnosis. The immune disease lesson covers the clinical categories.

Current College of American Pathologists guidance supports this initial laboratory approach when a monoclonal gammopathy is suspected:^{77 Keren DF, et al. Laboratory detection and initial diagnosis of monoclonal… 2022.}

1. Perform serum protein electrophoresis and serum free light chains.
2. If serum electrophoresis shows a monoclonal protein or the serum free light-chain ratio is abnormal, perform serum immunofixation or a validated equivalent.
3. For suspected amyloid light-chain amyloidosis, order serum protein electrophoresis, serum free light chains, serum immunofixation, and urine immunofixation together because the monoclonal protein may be very small.
4. Correlate all results because no single method detects every monoclonal protein.

Immunofixation electrophoresis separates proteins in parallel lanes and applies antisera to heavy and light chains. A restricted band that reacts with one heavy-chain reagent and one light-chain reagent identifies the isotype. Immunofixation is more sensitive than routine serum protein electrophoresis for small proteins, and it is primarily qualitative. Immunosubtraction can provide an equivalent identification step on validated capillary systems. Some laboratories use mass spectrometry to distinguish endogenous monoclonal proteins from therapeutic antibodies or to characterize difficult findings. If a restricted kappa or lambda band has no associated IgG, IgA, or IgM band, test for IgD and IgE before you report the protein as light-chain only.^{77 Keren DF, et al. Laboratory detection and initial diagnosis of monoclonal… 2022.}

Serum free light-chain testing measures unbound kappa and lambda light chains and calculates a kappa/lambda ratio. Renal impairment can raise both concentrations and change the expected ratio. Inflammation can also raise both. Assays differ in calibration and antibody reactivity, so the method's reference interval and renal interpretation rules apply. Antigen excess, nonlinearity, polymerization, and lot-to-lot behavior are additional analytical concerns.^{88 Mayo Clinic Laboratories. Immunoglobulin Free Light Chains, Serum.}

When a restricted peak can be measured reliably, densitometry estimates the M-protein concentration. Co-migration with transferrin, complement, or another protein can make that estimate inaccurate, especially in the beta region. A small peak may still be clinically important, and a large peak can be analytically difficult. Keep serial M-protein measurements on the same method whenever possible.

Urine protein testing distinguishes albumin-predominant glomerular loss, low-molecular-weight tubular protein loss, overflow of a filtered protein, and protein from postrenal inflammation or bleeding. The reagent-strip lesson covers the protein error of indicators, the strip's albumin preference, and confirmatory testing. Quantitative studies add concentration, creatinine normalization, and protein identity.

Automated urine total-protein assays commonly use dye-binding or turbidimetric reactions. Pyrogallol red-molybdate provides sensitivity at urine concentrations. Precipitation methods such as sulfosalicylic acid detect several protein classes but are affected by specimen turbidity, drugs, and radiographic contrast. Modified biuret and Folin-based methods also exist. These reactions have unequal responses to albumin, immunoglobulin light chains, and low-molecular-weight proteins, and no current higher-order system makes results from all manufacturers interchangeable.^{99 Miller WG, et al. Measuring urine albumin and total protein. 2026.}

Urine protein electrophoresis can support the mechanism of proteinuria. An albumin-dominant trace supports glomerular loss. Multiple low-molecular-weight bands support impaired tubular reabsorption. A restricted free light-chain band supports overflow proteinuria and requires immunofixation for identity. Mixed and postrenal patterns occur, so the trace does not establish the diagnosis by itself.^{1,71 Bishop ML, et al. Clinical Chemistry. 2023.7 Keren DF, et al. Laboratory detection and initial diagnosis of monoclonal… 2022.}

Kidney Disease: Improving Global Outcomes classifies albuminuria by ACR as A1 below 30 mg/g, A2 from 30 through 300 mg/g, and A3 above 300 mg/g. The equivalent SI decision points are below 3, from 3 through 30, and above 30 mg/mmol. Confirm a random ACR at or above 30 mg/g when appropriate, preferably with a subsequent first-morning void, because exercise, infection, menstruation, hydration, and day-to-day biological variation can affect a result.^{10,1110 Kidney Disease: Improving Global Outcomes… KDIGO 2024 clinical practice guideline for the evaluation and… 2024.11 Seegmiller JC, et al. Urine albumin measurements in clinical diagnostics. 2024.}

Suppose urine albumin is 66 mg/L and urine creatinine is 1.50 g/L.

ACR = 66 mg/L ÷ 1.50 g/L = 44 mg/g

The liters cancel, leaving milligrams of albumin per gram of creatinine. This result is in category A2. The category describes albumin excretion and is interpreted together with persistence, estimated glomerular filtration rate, and the clinical setting.

Suppose urine total protein is 52 mg/dL and urine creatinine is 104 mg/dL.

PCR = 52 ÷ 104 = 0.50 mg/mg = 500 mg/g

The local method and clinical pathway determine interpretation of PCR. Timed excretion calculations and collection checks are in Timed Urine and Renal Clearance Calculations.

Serum protein electrophoresis, serum immunofixation, and serum free light chains detect most monoclonal gammopathies more effectively than a urine screen alone. Urine immunofixation is used in selected settings, including suspected amyloid light-chain amyloidosis and some monoclonal kidney disorders. A 24-hour urine collection may be requested when quantitative urinary M-protein excretion is needed.^{77 Keren DF, et al. Laboratory detection and initial diagnosis of monoclonal… 2022.}

Cerebrospinal fluid contains far less protein than serum. Concentration varies with age and collection site. Lumbar fluid usually contains more protein than ventricular fluid. Blood contamination can add albumin and immunoglobulins. Quantitative methods therefore need a measuring interval and blank appropriate for low concentrations. Laboratories may use reflectance, turbidimetric, dye-binding, or immunochemical methods, with method- and age-specific reference intervals. Increased total protein is nonspecific and can accompany blood-CSF barrier dysfunction, inflammation or infection, hemorrhage, impaired CSF flow, tumor, or demyelinating injury. Exam-only intervals are listed separately in ASCP BOC Examination Reference Ranges.^{1,121 Bishop ML, et al. Clinical Chemistry. 2023.12 Mayo Clinic Laboratories. Protein, Total, Spinal Fluid.}

After concentration, a normal CSF electrophoretic pattern can show prealbumin, albumin, alpha-1, alpha-2, beta, and gamma proteins. Albumin remains the largest fraction. Beta-1 transferrin is the serum form. Beta-2 transferrin, also called the tau fraction in older electrophoresis terminology, is a carbohydrate-deficient transferrin form found in CSF and used as a marker of CSF in nasal, otic, or wound fluid. Immunoglobulins contribute to the gamma region. Fraction names and migration positions depend on the platform.^{1,131 Bishop ML, et al. Clinical Chemistry. 2023.13 ARUP Laboratories. Beta-2 Transferrin.}

The albumin quotient evaluates transfer of a liver-derived protein across the blood-CSF barrier. Convert the paired CSF and serum albumin concentrations to the same units before division:

albumin quotient (QAlb) = CSF albumin ÷ serum albumin

Suppose CSF albumin is 180 mg/L and serum albumin is 40,000 mg/L:

QAlb = 180 ÷ 40,000 = 0.0045 = 4.5 × 10⁻³

QAlb decision limits are age- and procedure-dependent. An increased quotient supports impaired blood-CSF barrier function or altered CSF flow. It does not identify a cause.

The IgG index compares the CSF-to-serum IgG ratio with the paired albumin ratio:

IgG index = (CSF IgG ÷ serum IgG) ÷ (CSF albumin ÷ serum albumin)

Suppose CSF IgG is 4.0 mg/dL, serum IgG 1,000 mg/dL, CSF albumin 20 mg/dL, and serum albumin 4.0 g/dL (4,000 mg/dL):

IgG index = (4.0 ÷ 1,000) ÷ (20 ÷ 4,000) = 0.004 ÷ 0.005 = 0.80

One current laboratory procedure uses 0.70 as its upper reference limit, so 0.80 is increased for that procedure. An increased index supports intrathecal IgG production. It does not establish a specific neurologic diagnosis.^{1414 Mayo Clinic Laboratories. Cerebrospinal Fluid IgG Index Profile, Serum and Spinal Fluid.}

Oligoclonal band testing uses isoelectric focusing followed by IgG-specific immunoblotting on paired CSF and serum. The serum comparison separates intrathecal bands from circulating bands that crossed into CSF.^{1515 Mayo Clinic Laboratories. Oligoclonal Banding, Serum and Spinal Fluid.}

Intrathecal IgG synthesis occurs in multiple sclerosis and in other inflammatory or infectious neurologic disorders. Myelin basic protein is a nonspecific marker of myelin injury. Alzheimer disease biomarker panels use procedure-specific combinations and decision limits. These results require the validated neurologic testing pathway. Cell counts, appearance, and infectious patterns are covered in Body Fluid Cell Counts and Cerebrospinal Fluid.

For every specimen type, report the protein pattern and the confirmatory test it requires. The pattern alone does not establish a diagnosis.