A. Electrophoresis is the migration of charged particles in some medium (either liquid or solid) when an electrical field is applied. Depending on the charge of the molecules, negatively charged particles migrate toward the positive electrode (anode), and positively charged particles migrate toward the negative electrode (cathode).
1. Migration rate depends on:
a. Charge of the molecule, which is directly proportional to rate of movement b. Size of the molecule, which is inversely proportional to rate of movement c. Electrical field, in which increased current increases migration rate
d. Ionic strength of buffer, in which increased ionic strength decreases migration rate
e. pH of buffer, in which decreased pH slows migration
f. Viscosity of supporting medium, which is inversely proportional to migration rate
g. System temperature, in which high temperature can denature protein and slow migration
2. Analytic electrophoretic procedures include protein electrophoresis and isoenzyme electrophoresis.
a. Protein electrophoresis
(1) The principle of protein electrophoresis
(a) Proteins are amphoteric (i.e., they can have positive or negative charge because of their acidic and basic side chains).
(b) The isoelectric point of protein is the pH at which a protein has no net charge.
(c) At pH 8.6, proteins are negatively charged and migrate toward the anode.
(d) If the buffer pH is higher than the isoelectric point of protein, the protein carries a negative charge and migrates toward the anode.
(2) The methodology of electrophoresis
(a) A support medium (agarose gel or cellulose acetate) is put in contact with the buffer.
(b) A sample is applied to the medium.
(c) A constant current or voltage is applied, and particles are allowed to migrate and separate.
(d) The support is fixed and stained to visualize protein bands.
b. Isoenzyme electrophoresis is typically performed to visualize the isoenzymes of some clinically relevant enzymes.
(1) The principle of isoenzyme electrophoresis is similar to that of protein elec- trophoresis because isoenzymes are proteins. The procedure is performed at a pH of 8.6, and the most negatively charged particles migrate toward the anode.
(2) The methodology involved in isoenzyme electrophoresis is similar to that used for protein electrophoresis.
B. Immunoassay is a chemical assay based on the highly specific and tight, noncovalent binding of antibodies to target molecules (antigens). Immunoassay is typically useful when the endogenous concentration of an analyte is very low.
1. Components in the immunoassay system include antigens and antibodies.
a. An antigen (ag) is a substance that can elicit an immune response (production of a specific antibody) when injected into an animal. The antigen is typically the analyte of interest.
b. An antibody (ab) is an immunoglobulin formed in response to a foreign substance (antigen). The antibody is the most important component of this system, because it determines the sensitivity (ability to detect small amounts) and specificity (the degree of uniqueness of the ag-ab reaction) of the procedure.
2. Immunochemical labels are necessary to detect the ag-ab reaction.
a. Enzyme labels are attached to the antibody. With the addition of a Chromagen, they allow the immunoassay results to be quantitated colorimetrically.
b. Fluorescent labels are attached to the antibody and are detected when a photon is released from a fluorescent molecule that is excited from its ground state to a higher state and then returns to the ground state. A drawback of this system lies with the autofluorescence of serum.
c. Chemiluminescent labels are compounds that undergo a chemical reaction and form an unstable derivative. Upon return to the ground state, they release energy in the form of visible light. The light is measured by a luminometer, and light intensity is related directly to the concentration of the reactants.
d. Radioisotope labels are compounds that have the same atomic number but different weights than the parent nuclide (e.g., 125I,14C). Radioisotopes decay to form a more stable isotope. In the process, they emit energy in the form of radiation (electromagnetic gamma rays) that can be detected and quantitated.
3. Immunoassay methodologies are based on the label attached to the antigen or antibody (Table 1–2).
C. Chromatography is a technique used to separate complex mixtures on the basis of different physical interactions between the individual compounds and the stationary phase of the system (a solid or a liquid - coated solid). The goal of this technique is to produce “fractions”
for quantitation.
1. Mechanisms of separation are based on the interactions of solutes with mobile and stationary phases.
Table 1–2 Methods of Immunoassay
Method Basis What is Labeled Use
Enzyme-linked immunosorbent assay (ELISA)
Enzyme-based Antigen in some methods;
antibody in others
Hormone testing
Enzyme-multiplied immunoassay technique (EIA, EMIT)
Enzyme-based Antigen Drug monitoring
Fluorescence-polarized immunoassay (FPIA)
Fluorescence-based Antigen Hormone testing
Surfactant/
albumin ratio Fluorescent
immunoassay (FIA)
Fluorescence-based Antigen (fluorescence is proportional to concentration of analyte)
Catecholamine testing
Radioimmunoassay (RIA)
Radiation-based Antigen Hormone testing;
drug monitoring Immunoradiometric
assay (IRMA)
Radiation-based Antibody Hormone testing;
drug monitoring
a. Adsorption chromatography (liquid-solid chromatography) is based on the competition between the sample and the mobile phase for binding sites on the solid (stationary) phase. Molecules that are most soluble in the mobile phase move fastest.
b. Partition chromatography (liquid-liquid) depends on the solubility of the solute in nonpolar (organic) or polar (aqueous) solvents.
c. Ion-exchange chromatography involves the separation of solutes by their size and the charge of the ionic species present. The stationary phase is a resin (can be cationic with free hydrogen ions or anionic with free hydroxyl ions present).
Anion- and cation-exchange resins mixed together are used to deionize water.
2. Chromatographic procedures
a. Thin-layer chromatography (TLC) is used as a semi-quantitative screening test.
A layer of absorbent material is coated on a plate of glass, and spots of samples are applied. The solvent is placed in a container and migrates up the thin layer by capillary action. Separation is achieved by any of the previously discussed modes (see above). Sample movement is compared with the standard, and fractions are calculated using retention factor (Rf), which is unique for specific compounds:
Retention factor (Rf)= distance component moves
total distance −distance solvent front moves b. High-performance liquid chromatography (HPLC) provides quantitative re-
sults. It is highly sensitive and specific. Apparatus consists of a pressure pump; a gel-filled column; a sample injector; a detector that monitors each component (e.g., spectrophotometers, amperometric detectors); and a recorder. Sample and solvent are pushed through the column, and the resulting eluent is read by the detector. The peaks that are detected and printed are specific and distinctive for each compound that is analyzed by HPLC.
c. Gas chromatography (GC) separates mixtures of volatile compounds. It can have a solid or liquid stationary phase. The setup is very similar to HPLC, except the solvent is a gas, the sample is vaporized, and detectors are thermal conduc- tivity or flame ionization. A special detector can be a mass spectrometer (MS), which measures the fragmentation patterns of ions (GCMS) and is used in drug identification. Gas chromatography is divided into two categories:
(1) Gas-solid chromatography, in which the absorbent is a solid material;
(2) Gas-liquid chromatography (most common method used in clinical labora- tories), in which the absorbing material is a liquid coated on a solid phase.