Immunoassays: ELISA, RIA and Luminex™

00:01 ELISA - Enzyme Linked Immunosorbent Assay, is a very, very commonly employed analytical method in immunology.

00:16 Plastic plates are coated with an antigen.

00:20 They can be coated with an antibody as well, but in this particular example we are coating the plates with an antigen.

00:27 Very often one will purchase already coated plates from a manufacturer.

00:36 The excess antigen is washed off.

00:41 Test antibody is then added.

00:44 So maybe this is an autoantigen that is coated on the plate.

00:48 And we want to discover whether a patient has autoantibodies against that autoantigen.

00:57 If they do, then they will bind, those autoantibodies will bind to the autoantigen.

01:04 Following a period of incubation, the plate is washed.

01:09 And then a ligand to the test antibody is added.

01:13 In this case the ligand is another antibody coupled to the enzyme horseradish peroxidase.

01:23 The plate is again washed and a substrate, a chromagen is added and a colored product is produced by the enzyme reaction. The amount of color is proportional to the amount of antibody bound to the antigen. In a sandwich immunoassay, first of all antibody is bound to the well of a microtitre plate, which is typically a 96 well plate.

01:56 Varying amounts of antigen are added.

02:00 Any unbound antigen is then removed by washing.

02:06 So for example, maybe we want to measure the amount of a particular substance in the serum of a patient.

02:17 This will be detected by a specific antibody.

02:25 Following washing, a labeled second antibody is added that is specific for a non-overlapping epitope of the antigen.

02:36 So let’s say we are trying to measure a cytokine.

02:42 The first antibody coating the plate would be for one epitope on that particular cytokine.

02:49 The other antibody would be for a different epitope, a different part of the same cytokine.

02:56 Labels include radioactive isotopes or an enzyme that causes a substrate to change color or emit light.

03:09 This would be in a chemiluminescence assay.

03:15 The unbound labeled second antibody is removed by washing.

03:21 And then the amount of second antibody bound is measured.

03:28 What is able to determine the amount of bound second antibody as a function of the concentration of antigen added with construction of a standard curve.

03:40 In the fluorescent microsphere Luminex assay, there are beads that are coated with particular antigens.

03:50 Patient’s serum is added to the coated beads and then incubated.

03:57 There is a reporter tag, for example fluorescently labeled anti-human IgG antibody added to detect the binding of the patient’s antibody to the antigen coating the bead.

04:12 These beads are then subject to a technique called flow cytometry, and signals that identify each bead are emitted and detected.

04:24 There is also a second signal that identifies the reporter tag.

04:31 The amount of signal is proportional to in this example, anti-hepatitis A antibody in the patient’s serum.

04:39 So in this particular example, we have beads coated with different antigens from Hepatitis A, B and C and one can detect that the patient has antibodies to Hepatitis A.