Dot-Immunobinding

Abstract

AbstractDot-immunobinding assay is a simple and sensitive method devised by authors Sumi S, Mathai A, and Radhakrishnan V.V. for examining antibodies in sera. This immunodiagnostic method is based on the dotting or marking a protein (antigen) as a spot on the nitrocellulose membrane. A procedure for the assay of antibodies in sera based on the antigen’s application as a spot to nitrocellulose filters is described. The method has the merit of being simpler in operation and more sensitive than comparable existing procedures. Either antibody or protein is spotted directly on nitrocellulose membrane disks. The sera to be tested for antibodies or protein is added to this disk and allowed to sit for some time. After rinsing the disk to remove the unbound antibody or protein, the second antibody attached to an enzyme is added and incubated. Following another wash, the substrate is added. One can directly show the occurrence of the antigen-antibody complex in nitrocellulose membrane disks. The formation of a purple to pink-colored, insoluble substrate product on the membrane is a positive result in the assay. This method permits the processing of several samples simultaneously. The complete operational procedure needs only 4–6 h. Dot-Iba is rapid, and the technical steps involved in the assay are much simpler than the other immunoassays such as enzyme-linked immunosorbent assay in detecting circulating antigen and antibody in clinical samples. Dot-immunobinding assay showed an overall sensitivity of 60% for tuberculous meningitis diagnosis in the hands of the authors of this assay. They also did not observe false-positive results. The authors showed that this method was highly specific and reproducible for diagnosing paucibacillary diseases such as extrapulmonary tuberculosis. Thus, this method described by Mathai and Radhakrishnan is best for laboratories in the developing world, with limitations in laboratory funds.KeywordsAntigenAntibodyDot-immunobinding assayDot-IbaNitrocellulose disksCirculating antibody