BMS100 LAB - ANTIGENS AND ANTIBODIES ("AIDS TEST")

 

A technique known as Enzyme-Linked ImmunoSorbant Assay (ELISA) is designed to detect small amounts of antigen (Ag) or antibody (Ab) in a complex liquid sample such as blood. The ELISA is the primary test for HIV ("AIDS virus") infection.

When an individual is infected with HIV, their immune system will produce anti-HIV antibodies, called primary antibodies or "1o Ab" in this procedure, that are specific for the HIV antigens with which the person was infected.

 

The ELISA can test for the presence of these specific antibodies in a person’s blood. If anti-HIV antibodies are present in a sample of blood then the person is considered HIV-positive.


ELISA

Part One (left)

1.      HIV antigen (HIV Ag) is added to small wells in a microtiter plate. The antigen “sticks” to the plastic surface.

2.      Donor serum (DS), which is essentially plasma from the person being tested, is added to the wells.  If anti-HIV antibodies (1o Ab) are present in the DS, these antibodies will bind to the HIV antigens that are attached to the plastic.

Part Two (center)

1.      A secondary antibody (2o Ab) is added.

o        2o Ab is capable of recognizing 1o Ab specifically and binding to it.

o        2o Ab has an enzyme called horseradish peroxidase (HRP) attached to it. This enzyme converts a colorless substrate (S) to a yellow product (P).

2.      The wells are "washed" ("rinsed") with a buffer solution.

o        If the primary antibody is present, the secondary antibody (with the enzyme) will be bound to it and will remain there during the wash.

o        Secondary antibodies that are not bound to primary antibody will be removed by the washing.

Part Three (right)

1.      Substrate (S) is added to the well and the reaction is given time to occur.

o        If the donor serum contained anti-HIV (1o) antibodies, the 2o Ab and the enzyme will be present and will cause the substrate to turn yellow (positive test result).

o        If the donor serum lacked anti-HIV antibodies, then the 2o Ab and enzyme will have been "washed out." The fluid will remain relatively colorless (negative test result).

·         The instructor will demonstrate the use of micropipettors.

o        All P1000 micropipettors should be set to “010.”

o        All P100 micropipettors should be set to “100.”

    To Draw Fluid Into the Micropipettor
  1. Depress the plunger of the micropipette to the first stop, and hold it in this position,
  2. Dip the tip into the fluid.
  3. Draw fluid into the tip by slowly releasing the plunger.
    To Expel Fluid Out Of the Micropipettor
  1. Touch the micropipette tip to the inside wall of the well into which you want to expel fluid.
  2. Slowly depress the plunger of the micropipette to the second stop to expel all fluid.

·         In order to avoid cross contamination of your samples, use a new micropipet tip whenever you change reagents.

·         All used micropipet tips must be ejected into the Biohazard receptacles.

·         Dispose of all liquids in the beaker marked "waste."

Procedure

1.      Label the microtiter plate with your initials and number the rows 1-4. You may find it easier to see if the plate is set on a white piece of paper.

2.      Obtain four disposable ("dispo") pipets and label them 1, 2, 3, and 4. These pipets will be used to "suck" fluid out of the wells as directed below.

3.      Obtain a disposable ("dispo") pipet and label it "PBS." This pipet will be used to add PBS to the wells as directed below.

4.      Use a micropipettor to add 100 microliters of HIV (viral) antigens to all wells. Incubate at room temperature for 5 minutes.

5.      Use the dispo pipet labeled "1" to remove as much fluid as possible from the Row 1 wells (place tip near bottom of well and "suck" out fluid). Repeat for the remaining rows using the dispo pipet with the appropriate number.

6.      Wash each well twice with phosphate buffered saline (PBS):

a.       Use the "PBS" dispo pipet to add fresh PBS to each well. Wells should be ¾ full - do not allow wells to overflow.

b.      Remove PBS from the wells with the dispo pipet of the appropriate number.

c.       Repeat for the second wash.

7.      Use a micropipettor to add 100 microliters of each of the following reagents to the wells. Use a fresh tip for each row.

a.       Row 1: Add the positive control (+) to each of the wells. The positive control contains anti-HIV antibodies and is used to verify that the test can reliably identify the antibodies (prevents "false negative").

b.      Row 2: Add the negative control (-), to each of the wells. The negative control does not contain anti-HIV antibodies and is used to verify that the test does not falsely indicate the presence of antibodies (prevents "false positive").

c.       Row 3: Add donor serum 1 (DS1) to each of the wells. "DS1" represents the blood of a subject being tested.

d.      Row 4: Add donor serum 2 (DS2) to each of the wells. "DS2" represents the blood of a second subject being tested.

8.      Incubate at 370C for 15 minutes.

9.      View the ELISA Animation.

o        The "Indirect Method" depicts the "AIDS Test" procedure you're about to follow.

o        The "Direct Method" is typical of some pregnancy tests and will be discussed later.

10.  Remove the reagents from each well with the dispo pipet of the corresponding number.

11.  Wash each well twice with phosphate buffered saline (PBS):

 .        Use the "PBS" dispo pipet to add fresh PBS to each well. Wells should be ¾ full - do not allow wells to overflow.

a.       Remove PBS from the wells with the dispo pipet of the appropriate number.

b.      Repeat for the second wash.

12.  Using a micropipettor with a new tip, add 100 microliters of secondary antibody (20 Ab) to each well.

13.  Incubate at 370C for 15 minutes. At the start of this incubation period ask the instructor to prepare the substrate.

14.  Remove the reagents from each well with the dispo pipet of the corresponding number.

15.  Wash each well twice with phosphate buffered saline (PBS):

 .        Use the "PBS" dispo pipet to add fresh PBS to each well. Wells should be ¾ full - do not allow wells to overflow.

a.       Remove PBS from the wells with the dispo pipet of the appropriate number.

b.      Repeat for the second wash.

16.  Using a micropipettor with a new tip, add 100 microliters of substrate to each well.

17.  Incubate at 370C for 10 minutes.

18.  Analyze your results. The presence of a yellow product constitutes a positive result.

19.  Dispose of all solid waste, including the test plates, in the Biohazard receptacles.

Spring 2010