Immunofixation Electrophoresis: Principle, Procedure and Interpretation

Principle

Immunofixation electrophoresis (IFE) is a two-stage procedure using agarose gel high-resolution electrophoresis in the first stage and immunoprecipitation in the second. Proteins are first separated by electrophoresis. Then, in the second stage, soluble antigen in the gel reacts with specific antibody, and the resultant antigen–antibody complexes become insoluble (as long as the antibody is in slight excess or near equivalency) and precipitate. The precipitation rate depends on the proportions of the reactants, temperature, salt concentration, and the solution’s pH. The unreacted proteins are removed by a washing step, and the antigen– antibody complex is visualized by staining. The bands in the individual lanes are compared with the precipitin bands obtained with a normal control serum. 

Sample Preparation

Fresh serum or urine is the specimen of choice. Evaporation of uncovered specimens may cause inaccurate results, so specimens must be handled carefully. Plasma should not be used, because the fibrinogen may adhere to the gel matrix, resulting in a band in all patterns across the gel. If storage is necessary, samples may be kept covered at 2°C to 8°C for up to 72 hours.

Reagents, Materials, and Equipment 

• Gel chamber to electrophorese, stain, destain, and then dry the gels IFE gels
• IFE protein fixative 
• Acid violet stain 
• Vials of anti-IgG, IgA, IgM kappa, and lambda antisera
• Tris-buffered saline 
• Citric acid destain 
• IFE templates (20) 
• Blotters and blotter combs
• Antisera template 
• REP prep 3100
• Gel block remover 
• 10 percent acetic acid
• 0.85 percent saline
• Power supply capable of providing at least 350 volts

Precautions

The gel contains barbital, which, in sufficient quantity, can be toxic. In addition, sodium azide is used as a preservative in certain reagents. To prevent the formation of toxic vapors, do not mix with acidic solutions. When discarding, always flush sink with copious amounts of water. This will prevent the formation of metallic azides, which, when highly concentrated in metal plumbing, are potentially explosive. In addition to purging pipes with water, plumbing should occasionally be decontaminated with 10 percent sodium hydroxide. The gels must be stored in the protective packaging in which they are shipped. Do not refrigerate or freeze. 

Procedure

1. Prepare all reagents, including the tris buffer, acid violet, and citric acid destain according to directions in the kit.
2. Plug the electrophoresis chamber into a power supply, and snap the electrophoresis lid into place on the chamber.
3. Dilute patient serum samples from 1:3 to 1:10, following instructions for the individual manufacturers.
4. Remove the gel from the gel pouch. Carefully detach from the plastic mold and discard the mold.
5. Dispense approximately 1 mL of REP prep onto the left side of the electrophoresis chamber.
6. Place the gel carefully into the electrophoresis chamber, making sure that any notches are aligned with pins on the chamber floor. Use a lint-free tissue to wipe around the edges of the gel backing to remove any excess REP prep. Make sure that no bubbles remain under the gel.
7. Using a blotter, gently blot the entire gel using slight fingertip pressure on the blotter. Remove the blotter.
8. Remove IFE templates from the package. Hold the template so that the small hole in the corner is toward the front right side of the chamber.
9. Carefully place the template on the gel, aligning the template slits with the marks on each side of the gel backing. The center hole in the template should align with the indention in the center of the gel.
10. Apply slight fingertip pressure to the template, making sure there are no bubbles under it. 
11. Apply the appropriate serum dilution to six template slits. Wait 2 minutes after the last sample application to allow proper absorption.
12. Gently blot the excess sample from the template. Then carefully remove the template.
13. Close the lid of the chamber. Set the power to 350 volts and start the power supply. Electrophorese the gel for 7 to 8 minutes, following instructions from the kit’s manufacturer. 
14. Turn off the power supply and remove the lid to the electrophoresis chamber.
15. Using the gel block remover, carefully remove the two gel blocks (one on each end of the gel). Use a lint-free tissue to wipe around the edges of the gel backing to remove any excess moisture.
16. Replace the electrophoresis lid with the drying lid. 
17. Holding the antisera template in the up position, place the template into the appropriate slots in the chamber.
18. Gently lower the antisera template onto the surface of the gel. No further pressure is needed.
19. Quickly pipette the fixative and antisera into the slots at the right end (anode) of each antisera channel in the template.
20. After 2 minutes (1 to 3 minutes is acceptable) incubation time, place one blotter comb into the same slots where the antisera has been applied. After 2 minutes, remove the blotter comb and the antisera template.
21. Gently blot the gel with a blotter and remove it. Place a different blotter on the surface of the gel. Place the antisera template on top of the blotter. After 5 minutes, remove the antisera template and blotter. Close the lid.
22. Dry the gel for 8 minutes by turning on only the chamber. After 8 minutes, turn the chamber off and remove the gel.
23. Fill a container with prepared stain. Fill another container with destain solution. Fill a third container with tris-buffered saline (TBS).
24. Place the gel in TBS and shake gently on a rotator for 10 minutes. Remove the gel from the TBS and allow it to drain on a blotter.
25. Place the gel into the staining dish containing the prepared stain. Leave it for 4 minutes. Remove the gel from the stain and allow it to drain on a blotter.
26. Destain the gel in two consecutive washes of destain solution. Use a gentle, alternately rocking and swirling technique. Allow the gel to remain in each wash for 1 minute. The gel background should be completely clear. Tap the gel to remove the excess destain solution.
27. Ensure that the chamber floor is clean. Replace the gel onto the chamber floor. Set a timer for 8 minutes, close the drying lid, and turn the chamber on.
28. Remove the gel from the chamber, and place it into the destain again for 1 minute. Tap the gel to remove excess destain solution.
29. Place the gel back into the chamber and dry for 5 minutes.
30. Turn off the chamber and remove the gel.

Results and Interpretation

The different lanes of patient serum are compared to the normal control. One lane will have all the serum protein bands stained, and the remaining five lanes will show individual reactions with antisera to heavy chains (anti-IgG, anti-IgA, or anti-IgM) and to light chains (anti-kappa and anti-lambda). The presence of abnormal bands is indicated by an increase in size and staining activity in a particular area. Polyclonal hypergammaglobulinemia is indicated by broad, dark bands in the gamma region. A monoclonal protein band produces a pattern that is narrow and dark. Conversely, a faint band in the gamma region indicates a hypogammaglobulinemia.

In the clinical laboratory, immunofixation electrophoresis is primarily used for the detection of monoclonal gammopathies. A monoclonal gammopathy is a primary disease state in which a single clone of plasma cells produces elevated levels of an immunoglobulin of a single class and type. Such immunoglobulins are referred to as monoclonal proteins, M-proteins,or paraproteins.In some cases they are indicative of a malignancy such as multiple myeloma or Waldenström’s macroglobulinemia. A monoclonal gammopathy will typically show a narrow dark band in one heavy-chain region and in one light-chain region. 

Differentiation must be made between polyclonal and monoclonal gammopathies, because polyclonal gammopathies are only a secondary disease state due to clinical disorders such as chronic liver diseases, collagen disorders, rheumatoid arthritis, and chronic infections. Polyclonal gammopathies are indicated by wide bands in one or more heavy- and light-chain regions. 

If there is a very high level of immunoglobulin in the patient sample, antigen excess may occur. This will result in staining of the margins, leaving the central area with  little demonstrable protein stain. In this case it will be necessary to adjust the protein content of the sample by dilution.