ELISA PART 2

ELISA

• Indirect sandwich assay– “Indirect” because the color reaction is

catalyzed by an enzyme linked to a secondary Ab (and not the primary Ab)

– “sandwich” because the antigen Ab we’re trying to detect is attached between the substrate attached Ab and the detecting primary Ab

GoatMouseRabbit

Substrate reacts with enzyme

Stop reaction

Identification of an “unknown” antibody

• You will be given a sample of a mouseantibody with one or more of the following serotypes– IgG1, IgG2a, IgG2b, IgG3, IgM, IgA

• Remember, antibodies are HIGHLY specific and can distinguish between the different types of IgG used

The primary Antibody

• Rabbit anti-mouse– Each specific antibody will only recognize

one type of mouse antibody

• The negative control well will contain a mix of all the primary antibodies used– What does this well test for?

The secondary Ab

• The secondary Ab recognizes ALL rabbit-derived Ab

• Also has an enzyme (peroxidase) covalently attached– Causes color change (the “development”

stage) in added substrate– Color change will only occur in the

presence of bound secondary Ab

Write up

• You must include a graph and a table– Your data from the graph will be summarized in

the table

• The dilution factor axis runs “backwards” from highest concentration to lowest– The titer is the reciprocal of the dilutions – Don’t forget the “control” on that axis

• In your diagrams, use proper nomenclature– “Rabbit anti-mouse-IgG2b IgG”

Sample data

02468

1012141618

1:01 1:04 1:16 1:64Dilution

Abso

rban

ce

controlunknownunknown 2

Threshold absorbance

Reminders

• ELISA mini-report due next week (10/6)

• RFLP due the following week (Thursday after midterm)