Sandwich ELISA and ELISPOT avoid a major problem of cytokine bioassays, namely, the ability of different cytokines to stimulate the same response in a bioassay. Bioassays must always be confirmed by inhibition of the response with neutralizing monoclonal antibodies specific for the cytokine. Another way of identifying cells actively producing a given cytokine is to stain them with a fluorescently tagged anti-cytokine monoclonal antibody, and then identify and count them by FACS (see Section A-23).
A quite different approach to detecting cytokine production is to determine the presence and amount of the relevant cytokine mRNA in stimulated T cells. This can be done for single cells by in situ hybridization and for cell populations by the reverse transcriptase–polymerase chain reaction (RT–PCR). Reverse transcriptase is an enzyme used by certain RNA viruses, such as HIV, to convert an RNA genome into a DNA copy, or cDNA. In RT–PCR, mRNA is isolated from cells and cDNA copies are made in vitro using reverse transcriptase. The desired cDNA is then selectively amplified by PCR by using sequence-specific primers. When the products of the reaction are subjected to electrophoresis on an agarose gel, the amplified DNA can be visualized as a band of a specific size. The amount of amplified cDNA sequence will be proportional to its representation in the mRNA; stimulated T cells actively producing a particular cytokine will produce large amounts of that particular mRNA and will thus give correspondingly large amounts of the selected cDNA on RT–PCR. The level of cytokine mRNA in the original tissue is usually determined by comparison with the outcome of RT–PCR on the mRNA produced by a so-called ‘housekeeping gene’ expressed by all cells.
A-30 Transfer of protective immunity.
Protective immunity to a pathogen may involve humoral immunity, cell-mediated immunity, or both. For studies in experimental animals such as inbred mice, the nature of protective immunity can be determined by transferring serum or lymphoid cells from an immunized donor animal to an unimmunized syngeneic recipient (that is, a genetically identical animal of the same inbred strain). If protection against infection can be conferred by the transfer of serum, the immunity is provided by circulating antibodies and is called humoral immunity. Transfer of immunity by antiserum or purified antibodies provides immediate protection against many pathogens and against toxins such as those of tetanus and snake venom (Fig. A.40). However, although protection is immediate, it is temporary, lasting only so long as the transferred antibodies remain active in the recipient’s body. This type of transfer is therefore called passive immunization, to distinguish it from active immunization with antigen, which can provide lasting immunity. A disadvantage of passive