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16.3.6: The EIA and Western Blot serologic tests for antibodies against the Human Immunodeficiency Virus (HIV)

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    In the case of the current HIV antibody tests, the patient's serum is mixed with various HIV antigens produced by recombinant DNA technology. If the person is seropositive (has repeated positive antigen-antibody tests), then HIV must be in that person's body stimulating antibody production. In other words, the person must be infected with HIV. The two most common tests currently used to detect antibodies against HIV are the enzyme immunoassay or EIA (also known as the enzyme-linked immunosorbent assay or ELISA) and the Western blot or WB. A person is considered seropositive for HIV infection only after an EIA screening test is repeatedly reactive and another test such as the WB has been performed to confirm the results.

    The EIA is less expensive, faster, and technically less complicated than the WB and is the procedure initially done as a screening test for HIV infection. The various EIA tests give a spectrophotometric reading of the amount of antibody binding to known HIV antigens.

    The EIA test kit contains plastic wells to which various HIV antigens have been adsorbed (see Fig. \(\PageIndex{1}\), step 1). The patient's serum is added to the wells and any antibodies present in the serum against HIV antigens will bind to the corresponding antigens in the wells (Fig. \(\PageIndex{1}\), step 2). The wells are then washed to remove all antibodies in the serum other than those bound to HIV antigens. Enzyme linked anti-human gamma globulin (anti HGG) antibodies are then added to the wells. These antibodies, made in another animal against the Fc portion of human antibodies by injecting the animal with human serum, have an enzyme chemically attached. They react with the human antibodies bound to the known HIV antigens (Fig. \(\PageIndex{1}\), step 3). The wells are then washed to remove any anti-HGG that has not bound to serum antibodies. A substrate specific for the enzyme is then added and the resulting enzyme-substrate reaction causes a color change in the wells (Fig. \(\PageIndex{1}\), step 4). If there are no antibodies in the patient's serum against HIV, there will be nothing for the enzyme-linked anti-HGG to bind to and it will be washed from the wells. When the substrate is added, there will be no enzyme present in the wells to give a color change.

    Fig. \(\PageIndex{1}\), Step 1
    Enzyme Immunoassay (EIA or ELISA) for HIV Antibodies

    Fig. \(\PageIndex{1}\), Step 2
    Enzyme Immunoassay (EIA or ELISA) for HIV Antibodies
    Illustration of known HIV antigens adsorbed to a test well. Illustration of the patient's antibodies binding to known HIV antigens in the test well.

    Known HIV antigens are adsorbed to test well.

    The patient's serum is added. If the serum contains antibodies against the known HIV antigens, they will bind to those antigens. All other antibodies are then washed from the well.
    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

    Fig. \(\PageIndex{1}\), Step 3
    Enzyme Immunoassay (EIA or ELISA) for HIV Antibodies

    Fig. \(\PageIndex{1}\), Step 4
    Enzyme Immunoassay (EIA or ELISA) for HIV Antibodies
    Illustration of enzyme-linked anti-human gamma globulin (anti-HGG) reacting with the Fc portion of the patient's antibodies bound to the known HIV antigens in the test well. Illustration of added substrate reacting with the enzyme attached to the anti-HGG producing a visible color change in the test well that can be measured with a spectrophotometer.
    Enzyme-linked anti-human gamma globulin (anti-HGG) is added to the well. (Anti-HGG is an antibody made by another animal against human IgG antibodies. An enzyme is then attached to the antibody.) The anti-HGG will with any human IgG antibodies bound to the adsorbed HIV antigens. All unbound anti-HGG is then washed from the well.

    The substrate for the enzyme attached to the anti-HGG is added to the well. The enzyme substrate reaction produces a visible color change which can be measured with a spectrophotometer. This shows that the patient's serum must have contained antibodies against the known HIV antigens. If there were no antibodies present then there would be no enzyme-linked anti-HGG in the well and no color-producing enzyme-substrate reaction.
    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0
    Animation

    If the initial EIA is reactive, it is automatically repeated to reduce the possibility that technical laboratory error caused the reactive result. If the EIA is still reactive, it is then confirmed by the Western blot test.

    The Western blot WB is the test most commonly used as a confirming test if the EIA is repeatedly positive. The WB is technically more complex to perform and interpret, is more time consuming, and is more expensive than the EIAs.

    With the WB, the various protein and glycoprotein antigens from HIV are separated according to their molecular weight by gel electrophoresis (a procedure that separates charged proteins in a gel by applying an electric field). Once separated, the various HIV antigens are transferred to a nitrocellulose strip (see Fig. \(\PageIndex{2}\), step 1 and Fig. \(\PageIndex{2}\), step 2). The patient's serum is then incubated with the strip and any HIV antibodies that are present will bind to the corresponding known HIV antigens on the strip (Fig. \(\PageIndex{2}\), step 3). Enzyme-linked anti-human gamma globulin (anti HGG) antibodies are then added to the strip. These antibodies, made in another animal against the Fc portion of human antibodies by injecting the animal with human serum, have an enzyme chemically attached. They react with the human antibodies bound to the known HIV antigens (Fig. \(\PageIndex{2}\), step 4). The strip is then washed to remove any anti-HGG that has not bound to serum antibodies. A substrate specific for the enzyme is then added and the resulting enzyme-substrate reaction causes a color change on the strip (Fig. \(\PageIndex{2}\), step 5). If there are no antibodies in the patient's serum against HIV, there will be nothing for the enzyme-linked anti-HGG to bind to and it will be washed from the strip. When the substrate is added, there will be no enzyme present on the strip to give a color change.

    Fig. \(\PageIndex{2}\), Step 1: Western Blot (WB) Test for HIV Antibodies

    Fig. \(\PageIndex{2}\), Step 2: Western Blot (WB) Test for HIV Antibodies

    Fig. \(\PageIndex{2}\), Step 3: Western Blot (WB) Test for HIV Antibodies
    Illustration of a western blot test strip with separated known HIV antigens. Illustration of a section of a western blot test strip containing known HIV antigen gp120. Illustration or the patient's antibodies reacting with known gp120 HIV antigen on the western blot test strip.

    Different known HIV antigens are separated on the WB test strip.

    Section of the strip with known HIV antigen gp120.

    The patient's serum is added. If the serum contains antibodies against any of the known HIV antigens, they will bind to those antigens on the strip. All other antibodies are then washed from the strip.
         
    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0

    Fig. 18, Step 4: Western Blot (WB) Test for HIV Antibodies

    Fig. 18, Step 5: Western Blot (WB) Test for HIV Antibodies
    Illustration of enzyme-linked anti-human gamma globulin (anti-HGG) reacting with the Fc portion of the patient's antibodies bound to the known gp120 HIV antigen on the western blot test strip. Illustration of added substrate reacting with the enzyme attached to the anti-HGG producing a visible color change on the gp120 area of the western blot test strip.
    Enzyme-linked anti-human gamma globulin (anti-HGG) is added to the well. (Anti-HGG is an antibody made by another animal against human IgG antibodies. An enzyme is then attached to the antibody.) The anti-HGG will with any human IgG antibodies bound to the adsorbed HIV antigens. All unbound anti-HGG is then washed from the strip. The substrate for the enzyme attached to the anti-HGG is added to the strip. The enzyme substrate reaction produces a visible color change. This shows that the patient's serum must have contained antibodies against the known HIV antigens on the strip where the reaction took place. If there were no antibodies present then there would be no enzyme-linked anti-HGG in the well and no color-producing enzyme-substrate reaction.
    Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0
    Animation

    It should be mentioned that all serologic tests give occasional false-positive and false-negative results. The most common cause of a false-negative HIV antibody test is when a person has been only recently infected with HIV and his or her body has not yet made sufficient quantities of antibodies to give a visible positive serologic test. It generally takes between 2 weeks and 3 months after a person is initially infected with HIV to convert to a positive HIV antibody test.

    Further Information

    For more information on HIV and AIDS, see the following Learning Object in my CourseArc lessons:

    Contributors and Attributions

    • Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)

    This page titled 16.3.6: The EIA and Western Blot serologic tests for antibodies against the Human Immunodeficiency Virus (HIV) is shared under a not declared license and was authored, remixed, and/or curated by Gary Kaiser.