Immunofixation

At a somewhat more basic level, both agarose and acrylamide gel systems have been used for direct immunofixation. In these gels, samples are electrophoresed and then immunofixed by either using stnps of cellulose acetate soaked in an antibody or the antibody is placed direcdy over the sample area of the gel. 

Particular uses of isoelectric focusing with transferrin-specific immunofixation detects asialotransferrin in nasopharyngeal or otogenous secretions to differentiate rhinorrhea and otorrhea (K9). 

Noll B, Hackler R, Pelzer M, Pelzer S, Nusser P, Maisch B, Schaefer JR, Steinmetz A (1999) Semi-automated rapid isoelectric focusing of apolipoproteins C from human plasma using PhastSystem and immunofixation. Clin Chem Lab Med 37 643-648... 

Capillary electrophoresis subsequent to immunosubtraction, which entails incubating the serum sample with beads coated with specific antisera followed by analysis of supernatants, is an attractive alternative to immunofixation and is amenable to automation (K36). 

Guidelines for clinical and laboratory evaluation of patients with monoclonal gammopathies have been proposed (K7). These proposals address in addition electrophoretic, immunofixation, and quantitative techniques for measurement of M protein, and also provide guidelines for serum viscosity and cryoglobulin measurements. 

In multiple myeloma, the neoplastic plasma cells secrete monoclonal proteins such as either IgG or IgA. Generally, either k or A. light chains are secreted. These M (monoclonal) proteins can be detected by serum and urine protein zone and immunofixation electrophoretic techniques, the latter providing better discrimination. The immunoglobulin levels exceed 25 g/L. Quantitation of immunoglobulins can be performed by rate nephelometry. 

D. F. Keren, High Resolution Electrophoresis and Immunofixation, Butterworths, London, 1987. 

Keren DF. High-resolution electrophoresis and immunofix-ation. Techniques and interpretation. Oxford Butter-worth-Heinemann, 1994. 

In clinical laboratory medicine, this procedure has been applied to the evaluation of human myeloma proteins. However, the method is being replaced by immunofixation (IF) electrophoresis, particularly for the study of protein antigens and their spHt products, and for evaluation of human myeloma proteins (see later discussion of IF electrophoresis). 

Alper CA, Johnson AM. Immunofixation electrophoresis A technique for the study of protein polymorphism. Vox Sang 1969 17 445-52. 

Keren DE High Resolution Electrophoresis and Immunofixation Techniques. Boston Butterworth-Heineman, 1994. 

There are several genetic variants or isotypes of Cp, but none of these, except for genetic deficiency (see previous discussion in this chapter), has known clinical significance. The most prevalent isotypes are CpA, CpB, and CpC, which are detectable by electrophoresis followed by either immunofix-ation or functional staining CpB is the common, or "wild, isotype. The gene encoding Cp is on chromosome 3q25. ... 

Electrophoretic variants, including C3F, may be confused with monoclonal immunoglobulins their identity can be ascertained by immunofixation using specific antiserum. 

Activation of C3 has been determined in the past by either immunofixation or two-dimensional, or crossed, Immunoelectrophoresis of plasma, with determination of the relative amounts of C3 and C3c. Currently, assays of fragments using antisera to neoaintigens (such as those on C3a) arfe preferred for this determination. For either type of assay, plasma samples must be obtained with precautions to prevent in vitro activation by plasmin, Cls, and leukocyte proteases The first few milliliters are discarded (or used for other purposes), then whole blood is collected through the same needle into a tube containing EDTA, Centrifugation should be performed as soon as possible and the separated plasma frozen below -40 C. 

Electrophoresis is used to study and measure the protein content of biological fluids (see Chapter 5). Types include serum protein electrophoresis using either cellulose acetate strip or agarose gel as the separation media, capillary electrophoresis (CE), immunofixation, and Western blotting. ... 

Immunofixation electrophoresis (IFE) is gradually replacing immunoelectrophoresis (lEP) for detection of. M-components because of its speed and ease of interpretation. Several procedures, including commercial Idts, are available for both IFE and lEP, Although these procedures may differ in detail, their principles are similar. 

Figure 20-10 Comparison of immunofixation electrophoresis (IFE) and immunoelectrophoresis (lEP) for two patients with monoclonal gammopathies. A, Patient specimen with an IgG (kappa, k) monoclonal protein as identified by IFE.The arrow indicates the position of monoclonal protein.After electrophoresis, each track except SPE is reacted with its respective antiserum, then all tracks are stained to visualize the respective protein bands. (SPE Chemically fixed serum protein electrophoresis IgG, IgA, IgM, k, and A, indicate antiserum used on each track.) B, Same specimen as in A, with proteins identified by lERThe arrow indicates the position of monoclonal protein. Norma control (C) and patient sera (S) are alternated. After electrophoresis, antiserum is added to each trough as indicated by the labels ig (polyvalent Ig antiserum), IgG, IgA, IgM, K, and A The antisera react with separated proteins in the specimens to form precipitates in the shape of arcs.The IgG and k arcs are shorter and thicker than those in the normal control, showing the presence of the IgG (k) monoclonal protein.The concentrations of IgA, IgM, and A,-iight chains are also reduced. C, Patient specimen with an IgA (lambda, A,) monoclonal protein identified by IFE procedure as described in A. D, Same specimen as in C with proteins identified by lEP as described in B.The abnormal IgA and A,-arcs for the patient specimen indicate an elevated concentration of a monoclonal IgA (A.) protein. Ait separations were performed using the Beckman-Coulter Paragon system.

Whicher JT, Higginson J, Riches PG, Radford S. Clinical applications of immunofixation Detection and quantification of complement activation. J Clin Pathol 1990 33 781-5. 

Serum immunofixation electrophoresis of two patients with monoclonal gammopathies. Pattern A represents IgG(/c) monoclonal gammopathy and pattern B represents IgA(/r) monoclonal gammopathy, as indicated by arrows. The procedure consists of serum protein electrophoresis (SPE) separation, reaction of each track with the exception of SPE with specific respective antiserum, followed by protein staining to make visible the respective bands. 

It should be noted that pseudomonoclonal bands appearing in the P-y region may be misidentified as authentic monoclonal gammopathy. Pseudomonoclonal bands occur in those serum specimens due to hemolysis or to the presence of fibrinogen due to inappropriate blood coagulation techniques. Serum immunofixation studies should clarify these problems. 

Levinson SS. Urine protein electrophoresis and immunofixation electrophoresis supplement one another in characterizing proteinuria. Ann Clin Lab Sci 2000 30 79-84. 

Harrison, H.H., Miller, K.L., Abu-Alfa, A., and Podlasek, S.J., 1993, Immunoglobulin clonality analysis Resolution of ambiguities in immunofixation electrophoresis results by high-resolution, two- dimensional electrophoretic analysis of paraprotein bands eluted from agarose gels. m. J. Clin. Pathol. 100 550-560. 

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