(Part of this paper was presented at the 14th European Congress of Clinical Microbiology and Infectious Diseases, Prague, Czech Republic, 1 to. In cases where WB is unable to identify the causative species, a cross-adsorption assay followed by a second WB on the resulting supernatant may be conclusive ( 11). WB detects both early occurring antibodies against nonspecific lipopolysaccharide (LPS) antigens and late occurring antibodies against specific protein antigens (SPAs) located in the rickettsial outer membrane ( 15). Poor species specificity is a major drawback, but this may be circumvented by using a multiple-antigen IFA where reactions to several species can be compared directly ( 4, 13). IFA, which can detect immunoglobulin G (IgG) and IgM antibodies, has a sensitivity rate ranging from 84 to 100% ( 1, 6, 13, 15). Several tests are available, but with the exception of immunofluorescence assay (IFA), Western blotting (WB), and cross-adsorption assay, most are not recommended ( 10, 11). Serology is the most widely used microbiological method for diagnosing rickettsial infections. ATBF typically occurs in clusters and has recently emerged as a common cause of acute febrile illness in international visitors to the region ( 2, 4, 8, 13, 14). The disease is caused by Rickettsia africae, a recently identified spotted fever group (SFG) rickettsia, and is transmitted by cattle ticks in large parts of rural sub-Saharan Africa ( 9). Mount samples in antifade mounting medium and image.African tick bite fever (ATBF) is a flu-like illness frequently accompanied by inoculation eschars, headache, and neck myalgia ( 7). Rinse samples three times with PBS, then perform 3 x 5 minute washes with PBS, protected from light.Ĩ. Note: You may need to use a higher concentration of directly labeled primary antibody than you would use for indirect immunofluorescence.ħ. Nuclear counterstains or labeled phalloidin may be included at this step. Incubate samples with directly labeled mouse primary antibody diluted in blocking buffer for 2 hours at room temperature. Rinse samples three times with PBS, then perform 3 x 5 minute washes with PBS, protected from light.Ħ. If staining human or animal tissue sections that may contain endogenous immunoglobulins, make sure the secondary antibodies also have minimal cross-reactivity to the tissue itself.ĥ. Note: Be sure to use highly cross-adsorbed secondary antibodies with minimal cross-reactivity for all antibody host species. Typically, fluorescent secondary conjugates are used at a final concentration of 1-2 ug/mL. Incubate samples with fluorescent anti-mouse and anti-rabbit secondary antibodies diluted in blocking buffer for 30 minutes to 2 hours at room temperature, protected from light. Rinse samples three times with PBS, and then perform 3 x 5 minute washes with PBS.Ĥ. Incubate samples with unlabeled mouse and rabbit primary antibodies diluted in blocking buffer at the manufacturer’s recommended concentration for 2 hours at room temperature or 4☌ overnight.ģ. Note: See our Protocols for Antibody-Based Detection for more information on fixation, permeabilization, and blocking.Ģ. Perform fixation, permeabilization, and blocking of your samples according to the preferred protocol for your primary antibodies. Protocol for combined direct and indirect immunofluorescenceġ.
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