Enzyme Linked Immunosorbent Assay

(ELISA)
 ELISA

• Enzyme Linked Immunosorbent Assay (ELISA)

• Digunakan untuk mendeteksi Antibodi dan antigen
• Sensitive, pg/mL
• Most commonly, ELISAs are performed in 96-
well (or 384-well) usually polystyrene microtiter
plates, which will passively bind antibodies and
proteins
 Less costly and safest.
 Easy visualization of results with high level of accuracy.
 Specific and highly sensitive assay that can detect protein at the
picomolar to nanomolar range.
 Easily automated for performance of large numbers of tests.
 Require minimal reagents.
 Qualitative detection or Quantitative measurement of either antigen or
antibody.
 Wells can be coated with antigens or antibodies.
 Can be done by personnel with only minimal training.
 Purified antigen (if you want to detect or quantify antibody).
 Purified antibody (if you want to detect or quantify antigen).
 Standard solutions (positive and negative controls).
 Sample to be tested.
 Micro-titer plates: plastic trays with small wells in which the assay is
done.
 Wash fluid (buffer).
 Enzyme-labeled antibody and enzyme substrate.
• ELISA reader (spectrophotometer) for quantitative measurements.
 Enzyme labels

• Enzyme labels are used to detect the binding of antigen-

antibody complex.
• It should have high specific reactivity.
• It should be easily coupled to antigen-antibody complex and
must stable.
• Enzymes used in labelling should not be normally present in
the patient samples.
• Examples of enzyme labels are Horse radish peroxidase,
Alkaline phosphatase, and Glucose oxidase.
 Stages in ELISA

1. The adsorption of either antigen or antibody to the micro-titer plate.

2. The addition of the test sample and subsequent reagents.
3. The incubation of reactants (formation of antigen-antibody complex).
4. The separation of bound and free reactants by washing.
5. The binding of enzyme to the target antigen or antibody.
6. The addition of substrate (production of a visible signal).
7. The visual or spectrophotometric reading of the assay.