WESTERN BLOT
Western blot or protein immunoblot is a technique to detect specific microbial proteins or patient antibodies to the proteins. This technique is a variation of Southern blot developed by Edwin Southern to detect DNA, and Northern blot developed for the detection of RNA. Although this technique has been replaced by other infectious diagnostic tests for most organisms, it is still used for diseases such as Lyme disease, prion-mediated Creutzfeldt-Jacob disease (CJD), and HIV. In the case of Lyme disease, the Western blot test is used to confirm an initial positive immunoassay result (Fig. 1). Microbial proteins (either purified or whole cell proteins) are denatured with a strong reducing agent and detergent and then separated by size on a polyacrylamide gel by electrophoresis. The proteins are then transferred to a filter paper sheet (nitrocellulose, nylon) by “blotting” (blocked with milk proteins to prevent nonspecific reactions) and then the blot is treated with the patient’s serum. After binding with the patient’s antibodies, the sheet is washed and stained with an enzyme-conjugated antibody to show the individual proteins recognized by the antibody.
Fig1. Western blot analysis. Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose (NC) paper, and incubated with antigen-specific antisera or a patient’s antisera (1 Ab) and then enzyme conjugated antihuman serum (2 Ab). Enzyme conversion of substrate identifies the antigen.
MATRIX-ASSISTED LASER DESORPTION/ IONIZATION–TIME OF FLIGHT
It is rare that a technology can fundamentally alter well established diagnostic testing methods, but that is precisely what the use of matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry has done. This technology is now widely used for the identification of bacteria, mycobacteria, yeasts, and molds, replacing biochemical and morphologic tests that were the corner stone of diagnostic microbiology for more than 100 years. The reason for this transformation is that the technology is highly accurate, technically simple to perform, rapid, and inexpensive. Bacterial and yeast colonies are removed from the agar culture plates, transferred to a target plate, dis solved with a strong organic acid (e.g., formic acid), mixed with an excess of ultraviolet (UV)-absorbing matrix, and dried on the target plates (Fig. 2). The dried preparations are exposed to laser pulses, resulting in energy transfer from the matrix to the nonvolatile protein molecules, with the desorption (removal) of the proteins into the gas phase. The ionized molecules are accelerated by electric potentials through a flight tube to the mass spectrometer, with separation of the proteins determined by their mass/charge ratio (m/z; z typically is 1), with smaller proteins moving more rapidly than larger proteins. The profile of proteins is compared with profiles of well-characterized organisms per mitting the identification of most organisms at the species or subspecies level. The entire process as described can be performed in minutes for bacteria and yeasts. Using MALDI TOF for the identification of mycobacteria and molds is only slightly more complex, resulting in highly accurate identifications in less than 1 hour. Selection of the matrix influences the specific biomarkers that are detected (e.g., proteins, phospholipids, cyclic lipopeptides) with α–cyano 4-hydroxy-cinnamic acid used preferentially for detection of protein biomarkers. The entire process is equivalent to identifying organisms by sequencing hundreds of genes because a single amino acid change will shift the protein profile. Applications of MALDI-TOF have recently been expanded for the detection of protein markers for antibiotic resistance and virulence, as well as the direct identification of some organisms in clinical specimens.
Fig2. Microorganism identification by matrix-assisted laser desorption ionization–time of flight–mass spectrometry (MALDI-TOF-MS). (From Wolk, D.M., Clark, A.E., 2018. MALDI-TOF mass spectrometry. Clin. Lab. Med. 38
