Dot-Immunobinding Assay (Dot-Iba) Protocol

Introduction of Dot-Iba Protocol

In most of the developing world, infectious diseases continue to remain as one of the major public health diseases. Most of these infectious diseases are potentially curable with appropriate antimicrobials. Any delay in diagnosis and treatment in patients will invariably lead to irreversible sequelae and complications. Development of newer techniques other than bacteriological methods has become not only essential but also relevant for the early and rapid diagnosis of paucibacillary conditions as in tuberculous meningitis. The newly developed assay should be more sensitive and also possess operational advantages over the existing diagnostic methods. The newly designed assay should be specific, cost-effective, and feasible for application in the developing world where there are constraints in laboratory resources.

Figure 1. NCM discs showing positive and negative results in Dot-Iba.

• Advantages of Dot-Iba

1. NCM discs have superior binding capacity than polystyrene microtiter plates.
2. The results can be read visually and there is no need of any expensive equipment such as microtiter ELISA readers.
3. The technical procedures involved in the assay are user friendly and laboratory personnel can be trained to undertake the assay.
4. Large number of specimens can be handled together.
5. Only limited laboratory space is required and there is no need for extraordinary instrumentation.
6. The NCM discs following the assay can be safely stored at 4 °C and it can be retained for review of previous results in the same patient.
7. The results can be made available within 6 h after the receipt of the specimen in the laboratory. Hence this assay is rapid and will be helpful in making decision at the bedside management of patients.
8. The Dot-Iba is best suited to laboratories in developing world where there is a definite limitation in the laboratory resources and technical expertise.

• Limitations of Dot-Iba

The Dot-Iba will yield negative results if the antigen concentration (as in specimens like CSF) is less than 50 ng/mL. So it is possible that the assay may yield false negative results in patients with tuberculous meningitis who received a course of anti-tuberculosis chemotherapy.

Notes of Dot-Iba Protocol

1. Circular discs should be cleanly cut with the help of a punching device. Discs with irregular edges and with wrinkles should not be used in the assay. NCM strips should be handled with a non- toothed and nonserrated forceps ("do not touch" with fingers).
2. Different concentrations (5–500 ng/mL) should be tried for the standardization procedure. In our experience, the dot-Iba gave positive results in all those NCM discs containing 50 ng/mL and above. In other words, the lowest detection limit in the assay is 50 ng/mL.
3. Do not release more than 5 μL of the antigen or antibody over the NCM disc. Also release the reagent steadily and uniformly over the NCM disc. Use a 5-μL micropipette with disposable tips for this purpose. During the release the reagent should not dribble outside the NCM disc.
4. The immunoreagents used in the assay such as antigen and antibodies must be stored in aliquots in optimal amounts and this should be used during the assay. This will also eliminate repeated effects of freezing and thawing. This will also enhance the shelf life of reagents.
5. As the result of the assay is interpreted by the visual examination of the NCM, the assay should be performed during daylight. However if the assay needs to be undertaken during the night, then a provision should be made to connect a light source to work bench.

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