How do I perform immunofixation electrophoresis (IFE) of urine?
1. A 25 ml patient urine sample is either filtered through a 0.45 µm syringe filter or centrifuged at 2,000 g to remove particulates and sediment that would interfere with the analysis. For electrophoretic analysis the protein concentration of the sample should be above 0.5 mg/ml. Since levels of BJP indicating a pathological condition can be as low as 0.01 mg/ml the sample must be concentrated. 5-10 ml of this clear urine is transferred to a BJP 5, 10 or 20 mL solvent absorption concentrator. The unit is left to stand until the sample has been concentrated 50-100 fold. 2. Concentrated patient urine (5 µl) is applied to the centre of each track on a specific IFE gel using an application mask. 3. Protein fixative and monospecific antibodies to IgG, IgA, IgM, Kappa and Lambda are applied to the gel. 4. During incubation, insoluble immune complexes are formed between the antisera and where present the immunoglobulins and light chains. 5. The gel is now washed, pressed, dried, and stained.
