Stefanie Dannenmann

ELISA Objective Enzyme-Linked ImmunoSorbent Assay (ELISA) are designed for detecting and quantitating substances such as peptides, proteins, antibodies and hormones. Principle An enzyme, that reacts with a colorless substrate to produce a colored product, is covalently linked to a specific antibody that recognizes a target antigen. Horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most commonly used enzymes for that assay. If the antigen is present, the antibody-enzyme complex will bind to it, and the enzyme component of the antibody-enzyme complex will catalyze the reaction generating the colored product. Therefore, the presence of the colored product indicates the presence of the antigen. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction. Such an enzyme-linked immunosorbent assay, which is rapid and convenient, can detect less than a nanogram of a protein. Indirect and sandwich ELISA There are two major types of ELISAs used: the indirect (Fig.1 A) and the sandwich (Fig. 1 B) ELISA. They are different in such a way that with the indirect ELISA an antibody can be detected and with the sandwich ELISA an antigen. The indirect ELISA is called “indirect” because a secondary antibody labeled with an enzyme is needed to detect an antibody that binds to a specific antigen. The sandwich ELISA is called “sandwich” because the analyte (antigen) to be measured is bound between two antibodies (capture and detection antibody), that bind to different epitopes on the antigen. Procedure

- Antigens (indirect ELISA) or antibodies (sandwich ELISA) are immobilized to the bottom of a polystyrene plate (96-well or 384-well plate)

- Blocking of all unbound sites to prevent false positive results - Sample that should be tested for the antibody (indirect ELISA) or antigen

(sandwich ELISA) is then added to the coated well and allowed to bind to the antigen or antibody respectively

- Enzyme-linked antibodies are added and allowed to react in the well - Unbound antibodies are removed by washing - Application of substrate - Enzymatic reaction suggests that the enzyme-linked antibody did bind and

that the sample contains the specific antibodies or antigens - The production of color is proportional to the amount of antibody or antigen

present

Variation Competitive inhibition assay: In that case antigen is labeled instead of the antibody. Unlabeled antibodies are attached to wells and a standard preparation of labeled antigen is bound to it. Unlabeled test samples are added and they compete with the labeled antigen for binding to the capture antibody. A decrease in signal indicates the presence of the antigen in the sample.

Stefanie Dannenmann

Fig.1: Indirect ELISA (A) and Sandwich ELISA (B) (Biochemistry, Fifth edition, Berg J.M., Tymoczko J.L., Stryer L., Clarke N.D., Fig. 4.35) Example Indirect ELISA: Basis of the test for HIV infection: In that case, viral core proteins are the antigens that are absorbed to the bottom of a well. After adding enzyme-linked antibodies an enzyme reaction indicates that the patient blood sample contains antibodies to the viral antigen. Sandwich ELISA: Detection of lipocalin 2 protein (from Flo T.H. et al., Nature 432 (2004), p. 917-921):

The figure shows the detection of lipocalin 2 in a sandwich ELISA. For detection of lipocalin 2 protein polyclonal antibodies were generated by immunizing rabbits with recombinant mouse lipocalin 2 protein. Affinity-purified antibody was used both as capture and detection (biotinylated) antibody in a sandwich ELISA. Panel b: Detection of the lipocalin 2 protein level in serum of BL/6 mice with and without Tlr4 receptor after LPS injection (black bares) compared with levels after PBS injection (white bars) . Panel c: Induced serum levels of lipocalin 2 in lipocalin 2 wild-type (open squares) and deficient mice (filled squares) after infection with the E.coli strain H9049.

c