RESUMO
This unit 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. One of these is a coating antibody, and the other is a derivatized detecting antibody. A support protocol describes chemical labeling of anti-cytokine monoclonal IgG antibodies with the NIP hapten group to produce the detecting antibody. Another support protocol describes a method for producing horseradish peroxidase (HRPO)-conjugated J4, a rat IgG1 anti-NIP monoclonal antibody that confers the anti-NIP specificity to the HRPO detection system used in the immunoenzymetric assay. The method described in this unit has allowed successful measurement of different cytokines in a wide variety of clinical samples including conditioned medium, serum and plasma, ascites, amniotic fluid, and bronchoalveolar lavage fluid.
