Immunoenzymetric Assay of Mouse and Human Cytokines Using NIPLabeled Anti Cytokine Antibodies

Detection and quantification of cytokines in clinical and biological samples has been made possible in large part by the use of specific and sensitive immunoassays employing anti-cytokine antibodies. Cytokine detection with antibody-based techniques is particularly useful because so many cytokines have overlapping, parallel, or counterregulatory bioactivities. It is therefore extremely difficult to distinguish them from one another and measure them in complex mixtures on the basis of biological activity alone.

The basic protocol describes a general method for immunoenzymetric assay of mouse and human cytokines. The technique is based on use of two anti-cytokine monoclonal antibodies (MAbs), each specific for a spatially distinct determinant on the cytokine (Abrams et al., 1992). One of these is a coating antibody, and the other is a detecting antibody derivatized with the haptenic group nitroiodophenyl (NIP). The NIP-labeled MAb is reacted with a horseradish peroxidase conjugate of a rat monoclonal anti-NIP antibody, and this in turn is detected with a chromogenic substrate. A schematic depiction of the assay reaction is presented in Figure 6.20.1.

The first support protocol describes chemical labeling of anti-cytokine monoclonal IgG antibodies with the NIP hapten group to produce the detecting antibody. The second support protocol describes a method for producing horseradish peroxidase (HRPO)-con-jugated J4, a rat IgG1 anti-NIP monoclonal antibody that confers the anti-NIP specificity to the HRPO detection system used in the basic protocol.

Figure 6.20.1 Schematic diagram of a general immunoenzymetric assay for cytokines. Assay uses a pair of anti-cytokine antibodies—a coating antibody bound to a microtiter plate and a detecting antibody derivatized with the hapten NIP—each of which recognizes a spatially distinct epitope on the cytokine molecule. The NIP-labeled antibody is then detected using a horseradish peroxidase (HRPO)-conjugated anti-NIP antibody, which is in turn visualized using a chromogenic substrate.

Figure 6.20.1 Schematic diagram of a general immunoenzymetric assay for cytokines. Assay uses a pair of anti-cytokine antibodies—a coating antibody bound to a microtiter plate and a detecting antibody derivatized with the hapten NIP—each of which recognizes a spatially distinct epitope on the cytokine molecule. The NIP-labeled antibody is then detected using a horseradish peroxidase (HRPO)-conjugated anti-NIP antibody, which is in turn visualized using a chromogenic substrate.

unit 6.20

Cytokines and Their Cellular Receptors

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