HIV

Introduction

The Human Immunodeficiency Virus (HIV) is a retrovirus, identified in 1983 as the etiological agent of the Acquired Immunodeficiency Syndrome (AIDS). AIDS is characterized by changes in the population of T-cell lymphocytes that play a key role in the immune defense system. In the infected individual, the virus causes a depletion of sub-population of T-cells, called T-helper cells, which leaves these patients susceptible to opportunistic infections and certain malignancies. The major routes of transmission are sexual contact, contamination by blood or blood products, and mother-to-newborn transmission.

The HIV virus consists of a genomic RNA molecule protected by a capsid and an envelop. The HIV envelop is the major target for humeral antibody response. The presence of the virus in the patients causes the immune system to elicit the production of anti-HIV antibodies, detection of which can be used as diagnostic tool. This particular assay system is a test device, which utilizes an immobilized specific binding receptor for analyzing an analyte in liquid samples derived from biological specimens. There are numerous immuno assays in which one component of an immunological pair (antigen/antibody) is detected or measured by complementary partner labeled with a tag which provide a detectable signal. The specificity of the analysis relies on the exquisite specificity of the antigen/antibody reaction. Obviously the specificity is determined by the specificity of the antigen used in the analysis.

Diagnosis

Both viruses (HIV-1 & HIV-2) produce antibodies in the infected individuals. Presence of HIV antibodies (IgM or IgG) in whole blood, serum, plasma or in any body fluid indicate the infection with HIV-1&2.

Detection of antibodies of HIV in the serological specimen is the most efficient and common way to determine that individual has been exposed to HIV.

Following test formats for the serological diagnosis of HIV are used

  • Latex / RBC agglutination based tests.
  • Immuno-gold conjugates based tests
  • Lateral Flow tests ( Line tests): NANO HIV 1 & 2 test kit
  • Flow through / immuno filtration tests (Dot tests): Product of JML & Endocrine.
  • ELISA based tests: Biochem immuno system, Korea Green Cross, Innogenetics etc.
  • Western blot tests
  • Polymerase chain reaction (PCR) tests

NANO HIV 1&2 test

Principle

The NANO HIV 1 & 2 is a rapid immunochromatographic screening test for the detection of antibodies to HIV 1 & 2 in human serum, plasma or whole blood. The test device contains test strip immobilized with recombinant antigens of HIV1 at test line (1), HIV 2 recombinant antigen at test line (2) and control line reagent at (C) on nitrocellulose membrane and dried gold conjugate sensitized with HIV antigens and anti control line.

The specimen is applied to the sample well followed by addition of running buffer. The buffer facilitates the lateral flow of the sample and gold conjugate as well as promoting the immunologic binding of antibodies and antigen, if present in patient's specimen. The HIV antibodies present in the patient's specimen binds to the gold conjugate and make a immune complex. The complex migrates on the nitrocellulose membrane and is captured by the antigens immobilized in the Test line zone (1 & 2) and produces pink/purple band with varying intensity, proportional to the HIV 1& 2 antibodies in the sample. In the absence of HIV 1/2 antibodies, there is no pink/purple band in the TEST line zone (1&2). The liquid continues to migrate along the membrane and produces another pink/purple band in the CONTROL line zone (C), demonstrating that the reagents are functioning properly and validates the result.

Immobilization of proteins on nitro cellulose membrane

  • GP-41, GP 120 & p24 on test line 1 for capturing of HIV-1 antibodies.
  • GP-36 on test line 2 for capturing of HIV-2 antibodies.

Gold conjugate

Colloidal gold sensitized with recombinant proteins of HIV-1 & HIV-2 applied below the sample addition pad in form of dried pad & laminated with Nitro cellulose membrane of the test strip. It dissolves on addition of specimen & running buffer. No need to add separately as required in flow through tests.

Sensitivity: 99.5%

Specificity: 99.7%

Test Procedure

  • Allow the kit components & test specimen to come at room temperature if stored in refrigerator.
  • Remove as many as test devices required for the testing from pouch by tearing along notch provided.
  • Lay down the test device on flat surface and mention patient's ID number.
  • Dispense one drop (20µl) of serum, plasma or whole blood using provided dropper or micro pipette into sample well (S).
  • Add 2-3 drops of running buffer using provided dropper bottle or dropper into sample well (S).
  • Read the result in result window within 5-20 minutes of specimen and running buffer addition.

Note: Used test devices, droppers, gloves, blood / serum collection tubes, swab, lancets etc. should be discarded as per norms of Biomedical waste management.

Interpretation

Positive: The appearance of pink/purple line at '1' in addition to control line at 'C' indicates that specimen contains antibodies of HIV-1. The appearance of test line at '2' in addition to control line at 'C' indicates that specimen contains antibodies of HIV-2. The appearance of test line at '1' & '2' in addition to control line at 'C' indicates that specimen contains antibodies of HIV-1 & 2. The intensity of lines at '1', '2' and 'C' may vary with each other.

Negative: The appearance of no pink/purple lines at '1' & '2' and only one pink/purple line at 'C' indicates that specimen does not contain antibodies of HIV 1 & 2 and specimen should be considered negative. A negative result after 20 minutes of incubation indicates that there is no detectable level of antibodies in patient's specimen.

Invalid: If there is no distinct pink/purple line visible at 'C', the test is invalid and it is recommended to repeat the test again.

NANO HIV 1&2 Vs Flow through dot tests

Characteristics Flow through test NANO HIV1&2 test
Test principle Test principle Flow through/ immuno filtration. Does not perform well if specimen is slightly turbid. WHOLE BLOOD can't be used. Lateral flow / immuno chromatographic. Perform well even if specimen is slightly turbid. WHOLE BLOOD can be used.
Revealing proteins Recombinant antigens immobilized on membrane but conjugate sensitized with Protein-A, which detects major part of IgG antibodies. Scientifically less sensitive and early infection may not be diagnosed.
Enclosed : Annexure 1
Recombinant antigens immobilized on membrane and conjugate sensitized with HIV recombinant antigens, which detects all types of HIV antibodies. Early infection HIV shall be diagnosed.
Choice of specimen Only serum or plasma, separation process time consuming & risk of exposure with HIV infective specimen is for longer time. Whole blood, serum or plasma. Whole blood process saves time and risk of exposure with HIV infective specimen is for lesser time.
Procedural steps More than one (Probably 5 steps) Only two ( Addition of specimen & running buffer).
Storage Refrigeration must (Recommended 2-8oC) Refrigeration optional (Recommended 4-30oC)
Shelf life Maximum 12 months from date of manufacturing 24 months from date of manufacturing
Important Kit components Test devices, wash buffer & Gold conjugate Test devices & running buffer. Inbuilt gold conjugated in test strip.
Sero diagnosis IgG1, IgG2, IgG4, antibodies IgG1, IgG2, IgG3, IgG4 , IgM & IgA antibodies
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