The various techniques just mentioned are based on different properties of protein molecules (e.g., chemical, dye-binding, electrical charge, antibody binding sites). Therefore, the different techniques may not produce identical values for all protein fractions or for individual proteins. Added to this are alterations in proteins from disease or genetic abnormalities, different interfering substances or medications, and technical problems unique or important for each technique or method. For example, the serum albumin value by electrophoresis may be about 0.5 gm/100 ml (5 g/L) less than the value determined by the usual chemical method. This is fortunately not often very important as long as the same laboratory performs the tests on the same patient and provides its own reference range. The differences in technique and methodology are magnified when the quantity of protein being assayed is small, such as in urine or spinal fluid. Since even modifications of the same method can produce slightly but significantly different results, reference values should be obtained by the individual laboratory for its own particular procedure.
Tag: Assay Methods
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Serum Protein Assay Methods
There are many widely used techniques for fractionating the serum proteins. “Salting out” by differential chemical solubility yields rough separation into albumin and globulin. Cohn devised a more complicated chemical fractionation method by which certain parts of the protein spectrum may be separated from one another in a large-scale industrial-type procedure. Albumin is most often assayed by a chemical method (biuret) that reacts with nitrogen atoms, or with a dye (such as bromcresol green or bromcresol purple) that preferentially binds to albumin. The ultracentrifuge has been used to study some of the subgroups of the globulins. This is possible because the sedimentation rate at high speeds depends on the molecular size and shape, the type of solvent used to suspend the protein, and the force of centrifugation. The velocity of any particular class of globulins under standard conditions depends primarily on molecular size and is known as the “Svedberg number”; the most common classes of globulins are designated as 7S, 19S, and 22S. Electrophoresis separates molecules by the electrical charge of certain structural atomic configurations and is able to subdivide the globulins, but only into groups rather than into individual proteins. Serum protein nomenclature derived from electrophoresis subgroups the serum globulins into alpha, beta, and gamma, corresponding to electrophoretic mobility. Using antibodies against antigens on the protein molecule, immunoassay (including radial immunodiffusion, Laurell “rocket” electroimmunodiffusion, immunonephelometry, immunofluorometry, and radioimmunoassay, among others) is another technique for measuring serum proteins that is assuming great importance. Immunoassay techniques quantitate individual proteins rather than protein groups and in general produce reliable results with excellent sensitivity and specificity. Immunoelectrophoresis or similar techniques such as immunofixation goes one step beyond immunoassay and separates some of the individual globulin molecules into structural components or into subclasses. Immunoelectrophoresis also can detect abnormal proteins that either differ structurally from normal proteins or are produced with different proportions of structural components.