LIFE公司的ELISA Protocol (General Guidelines)

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Introduction
Sandwich enzyme-linked immunosorbent assays (ELISAs) involve attachment of a capture antibody to a solid phase support. Samples containing known or unknown antigen are then added in a matrix or buffer that will minimize attachment to the solid phase. An enzyme-labeled antibody is then added for detection.

The ELISA method is a benchmark for quantitation of pathological antigens and there are indeed many variations to this method. ELISAs are adaptable to high-throughput screening because results are rapid, consistent and relatively easy to analyze. The best results have been obtained with the sandwich format, utilizing highly purified, prematched capture and detector antibodies. The resulting signal provides data which is very sensitive and highly specific. Thus, Invitrogen offers a large menu of sandwich ELISA and phosphoELISA™ kits. ELISA and phosphoELISA™ kits allow for quantitative measurements of proteins outside and inside the cell, respectively. For intracellular proteins for signaling studies, we offer phosphoELISA™ kits for measurement of total and phosphorylation, modification, or cleavage site–specific proteins.

The  phosphoELISA™ kit protocol (Figure 1) begins with a monoclonal antibody specific for a protein of interest (regardless of phosphorylation state) coated onto the wells of microtiter strips. Samples, including a standard containing protein of interest, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the protein antigen binds to the immobilized (capture) antibody, much like an immunoprecipitation. After washing, a rabbit antibody specific for total protein or protein phosphorylated at a specific residue (e.g., Akt [pS473]) is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized protein captured during the first incubation. After removal of excess detection antibody, a horseradish peroxidase–labeled anti–rabbit IgG antibody (anti-rabbit IgG-HRP) is added. This binds to the detection antibody to complete the four-member sandwich. After a third incubation and washing to remove all the excess anti-rabbit IgG-HRP, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of total or phosphorylated protein present in the original specimen. Invitrogen® phosphoELISA™ kits have been designed to enable the specific detection of phosphorylation of key signaling molecules, offer great specificity, and correlate well to western blot data.

The assay procedure for ELISA (Figure 2) and phosphoELISA™ kits are similar with only a few minor differences.  While the detector antibody for ELISA kits is biotin-labeled and is followed by incubation with streptavidin- HRP, the detector antibody for phosphoELISA™ kits is a rabbit polyclonal antibody and is followed by incubation with anti-rabbit IgG-HRP.

Figure 1. Schematic of phosphoELISA™ protocol.	Figure 2. Schematic of ELISA protocol.

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