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3. Monoclonal antibodies - ELISA Link to the Medical Research Council web site
ELISA - technical genius
ELISA (enzyme-linked immunosorbent assay) is a highly sensitive diagnostic technique which is used routinely in many laboratories. It involves the specific recognition by a monoclonal antibody (mAb) of a particular molecule (antigen) in a mixture. The technique is both qualitative and quantitative and involves the following procedure (see Figure 8):
Figure 8. ELISA procedure.

Step 1. The sample material (including the test antigen) is bound to a plastic plate.

Step 2. Monoclonal antibody (mAb) is then applied and left to bind to its specific antigen.

Step 3. The plate is then washed with buffer solution.

Step 4. A second antibody which will recognise and bind to the mAb-antigen complexes is then applied. Attached to this second antibody is an enzyme which can convert its substrate to a coloured product.

Step 5. The enzyme's substrate is added to the ELISA plates which allows both qualitative and quantitative assays of the test antigen to be obtained.

Question 4

a) Explain the basis for the specificity of a mAb.

b) Why is the plate washed with buffer solution (Step 3)?

c) What is meant by a 'qualitative assay of the test antigen' (Step 5)?


d) Explain how the amount of test antigen in the sample (quantitative assay) can be established.

Screening for disease
Characteristic of individuals infected with the Human Immunodeficiency Virus (HIV) is the presence in the body fluids of antibodies against the virus - 'anti-HIV antibodies'. The ELISA technique may beused to screen blood samples for the presence of anti-HIV antibodies.

The basic procedure is as outlined at the start of this activity, but specific details include:

  • The ELISA plate is prepared by binding synthetic and engineered HIV envelope proteins to the wells
  • The blood sample from the patient is washed over the plate.
  • The enzyme bound to the second antibody is horseradish peroxidase which converts its substrate into a blue product.
  • The enzyme-substrate reaction is halted by addition of sulphuric acid, which has the effect of converting the product from blue to yellow
  • An automated system is used to measure and record the optical density (using a wavelength of 450nm) of all wells
  • An optical density reading of less than 0.2 indicates that the sample is negative for anti-HIV antibodies; a positive reading is normally between 2.0 and 3.0.
Replicate wells (1-4):
Optical density reading
1 2 3 4 Sample
A 2.10 2.20 2.15 2.05 Patient A
B 2.98 2.96 2.94 2.98 Patient B
C 0.16 0.12 0.12 0.13 Patient C
D 3.00 2.97 2.99 2.96 Sample D
E 0.14 0.15 0.15 0.13 Sample E
Question 5

Blood samples from patients A, B and C were screened for anti–HIV antibodies using an ELISA. The results are shown on the left.

a) Explain the results shown in the table on the left.

b) What are the other samples (D and E in the table) which have been included in the assay?

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