The Western Blot (WB), or western immunoblot (WB), is a laboratory technique used to detect antibody to specific proteins by means of an indirect immunoassay. WB is frequently performed in conjunction with other test methodologies and is very useful in providing additional test specificity, for example in the case of testing for HIV or Lyme disease.
The enzyme-linked immunoelectrotransfer blot technique (Western Blot) is performed in three stages. The first stage utilizes gel electrophoresis to separate native or denatured proteins from a whole organism by their length, molecular weight, or 3D structure. The second stage involves the transfer of the electrophoresed proteins in the gel onto a nitrocellulose membrane. The third stage involves reacting patient specimen with the electrophoresed proteins contained on the nitrocellulose membrane. Positive reactions are developed by an enzyme immunoassay.
The Imugen laboratory utilizes IgG WB testing as part of the Lyme Antibody Analysis. Although IgM WB can be performed, the inherent test sensitivity and specificity are less well established than IgG WB. B. burgdorferi spirochetes are derived from cell culture. The laboratory typically employs two different strains – G39/40 is a “native” or wild strain originally isolated from a patient in CT; strain 49736 is a tick isolate from the upper Midwest which lacks the gene for OspA (the Lyme vaccine, no longer on the market, contained purified recombinant OspA). Employing these two distinct strains potentially improves test sensitivity. Furthermore, comparing WB’s with these two strains distinguishes patients who have received Lyme vaccine in the past from those with real exposure to B. burgdorferi, avoiding potentially false positive test results in those who have received Lyme vaccine.
The organisms are sonicated and the proteins are separated according to their molecular weight by gel electrophoresis and transferred onto nitrocellulose membranes. The nitrocellulose membranes are cut into individual test strips. The patient’s specimen is incubated with a test strip and if specific antibodies to B. burgdorferi proteins are present, they will bind to the proteins on the membrane. To detect bound protein antigens an enzyme conjugated IgG antibody is added and will bind to the antigen complexed with patient antibody. When a substrate solution is added, the bound conjugate will catalyze the substrate to form a precipitate at the site of the antigen antibody complexes. Discrete protein bands appear corresponding to specific molecular weights expressed as kilodaltons (kDa).
The more developed the antibody response to B. burgdorferi is will be reflected in the protein banding pattern observed by Western Blot. Typically, if the patient has developed antibodies to a significant number of proteins this will indicate an infection of greater duration. The relationship of disease stage to band number and intensity has been correlated and can be beneficial in the patient evaluation. The Imugen laboratory reports all observed bands on each of these two strains, not just the limited 10 bands initially suggested by some in the past. In late stage infections, important information is contained in the number of specific bands, and this is especially critical in paired specimens when there is a question of ongoing infection, re-infection, or relapse.
The WB technique can successfully be applied to other infectious agents, such as Babesia. The laboratory uses a native B. microti antigen in the preparation of Western Blot membranes. Both IgM and IgG WB tests are performed for the testing of B. microti infection. The IgM WB testing has the capability of detecting an early response in the course of B. microti infection.
The staff at the Imugen laboratory is highly trained in performing and interpreting these assays, has decades of experience in analyzing these test results in the context of various clinical situations, and is available to assist healthcare providers in interpreting tests and answering questions about the detection of tick-borne infections.