The HIV Dot Test Of AIDS Patients

HIV I and HIV II are the causal agent of AIDS (acquired immuno-deficiency syndrome). After entering in the blood of humans, HIV disrupts the immune system leading to multiple complex diseases called ‘disease syndrome’. Now-a-days, the HIV kits are available in market. The HIV DOT kit has in-built procedural control combined test for HIV I and HIV II. The HIV DOT test is a rapid (requires 3-5 minutes) visual screening test in human serum or plasma. It is an accurate immunoassay of HIV I and HIV II antibodies in human serum/plasma. However, it does not require any instrument, equipment, electricity, incubation and precise liquid additions. All reagents are ready to be used in the kit. This test is quite cheap, absolutely safe and extremely reliable. Each reagent is individually packed in dropper vials eliminating cross contamination.

The test device is such that it contains a porous membrane having two visible circles. The membrane is supported with absorbent. The HIV peptide antigens (toxin) are immobilised onto a porous membrane. The test sample of a patient and solutions can pass through the membrane and absorbed into the underlying absorbent. The order of working is as below:

  • Colloidal gold conjugate solution
  • Protein A
  • HIV antibodies
  • (Ag Ag Ag)


  • Suspected HIV patient
  • A clean and dry sterile vial
  • Centrifuge
  • The test device
  • Buffer solution
  • Wash solution
  • Colloidal gold conjugate
  • Positive control (no risk of infection synthetic recombinant peptide used)
  • Sample dropper (disposable pipette)


  1. Collect blood from the suspected HIV patient(s) in a clean, dry and sterile glass vial and
  2. allow to clot.
  3. Centrifuge the blood at room temperature to separate the serum. Assay the serum immediately. However, it may be stored at 2-8ºC.
  4. Bring all the solution, test device and sample to room temperature before the start of the test.
  5. Add 2 drops of buffer solution into the inner circle of the device (Fig. 15.5) and wait for a few second to get it soaked.
  6. Add 1 drop of the patient’s serum/plasma with the help of a dropper provided with the kit.
  7. Wait to get the serum soaked by the membrane.
  8. Add 2 drops of buffer solution and allow it to soak in.
  9. Again add 2 drops of wash solution and wait to soak in the membrane.
  10. Add 2 drops of colloidal gold conjugate solution and wait to soak in.
  11. Finally, add 3 drops of wash solution and record the result.


If the patient’s serum contains the HIV antibodies, it will bind to the HIV peptide antigens (toxin). The conjugate complex will bind to the Fc portion of the HIV antibodies to give a distinct red colour against a white background. Therefore, the positive result will show two visible dots in the inner circle. However, if only one dot is visible, it shows that the patient’s serum does not contain detectable HIV antibodies giving the negative result. On the other hand if none of the dots appear, it shows the procedural error or deterioration of reagents. In such cases, re-test the specimen with a new test device. 

For the assessment of positive procedure a control dot appears in the inner circle. It is because a rabbit IgG is immobilised on the membrane surface. This control verifies that the test reagents (colloidal gold conjugate, wash buffer and the test device) are functioning well and reagents have been added in the correct order.


Practical Microbiology

Gaurav Singh

Editor in Chief Medical Microbiology & Recombinant DNA Technology (RDT) Labs - RDT Labs Magazine

Leave a Reply