How to Determine Antibodies

The ELISA test, or enzyme-linked immunosorbent assay, is the most common commercially used test to screen for the presence of antibodies. This test is used to screen for drug use, pregnancy, AIDS and hepatitis, among other conditions. This test is popular because of the ease of use and low cost.

Things You'll Need

• Serum sample
• ELISA plate
• Antigens that conjugate the antibody being tested for
• Enzyme linked antibodies or secondary antibodies
• Enzyme linked color change indicator
• Paper towels
• Goggles

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Instructions

• 1

  Put a known antigen onto the surface of several of the wells.

  Cover each well with 50 microliters of IgG fluid. Do not disturb the wells until 5 minutes has passed. Rinse the wells with tap water. Repeat the rinsing process five times. Tap the well on a paper towel to dry it. Do not wipe the wells.

• 2

  Add samples of body fluid, called serum, to the plates. These body fluid samples may contain the conjugate and competitor solution to the wells. This will bind if the antibodies are present. The serum samples must be diluted so that they will conjugate to the antigen applied in step 1.

• 3

  Add a protein, such as casein, to the wells. This will prevent other proteins that aren't being tested for from adhering to the plate.

• 4

  Wash the plate by rinsing with tap water poured gently over the wells. Next, tap the side of the plate on a paper towel to remove excess water. Repeat the washing process four more times. Do not wipe the wells. Add the antibody that matches the one being tested for to the wells.

• 5

  Wash the plate by rinsing with tap water poured gently over the wells. Next, tap the side of the plate on a paper towel to remove excess water. Do not wipe the wells. Repeat the washing process four more times. This washing process will remove any antigen that isn't bound from the well. The wells now contain only antibody-antigen complexes that are being tested for. Wash the plate. This will remove any unbound antibody enzyme conjugates.

• 6

  Add secondary antibodies, also called anti-antibodies, to the wells. These will stick to the detection antibodies. The secondary antibodies are conjugated to the color change indicator, and to the antigen that was added to the well in step 1.

• 7

  Wash the plate by rinsing with tap water poured gently over the wells. Next, tap the side of the plate on a paper towel to remove excess water. Do not wipe the wells. Repeat the washing process four additional times. This will remove extra enzyme linked antibodies that aren't bound.

• 8

  Apply an enzyme linked color change indicator to the plate.

  This will cause the wells that have the antibodies being tested for to change color.

• 9

  Observe the wells of the plate for a color change, which will indicate the presence of the antibody in the body fluid sample.