Porcine lymphocyte gamma interferon responses to mitogenic stimuli monitored by a direct immunoassay
M Gonzalez-Juarrero1, C Mebus, A E Garmendia
1Plum Island Animal Disease Center, USDA, Greenport, NY 11944.
Insights
A new radioimmunoassay (RIA) was developed to measure porcine interferon gamma (PoIFNγ). This assay accurately quantifies PoIFNγ levels in biological fluids, offering a valuable alternative to traditional bioassays for immune response studies.
Area of Science:
- Immunology
- Biochemistry
Background:
- Porcine interferon gamma (PoIFNγ) plays a crucial role in immune responses.
- Accurate quantification of PoIFNγ is essential for understanding cellular immunity in pigs.
- Existing bioassays for IFNγ can be time-consuming and less sensitive.
Purpose of the Study:
- To develop and validate a direct, rapid, and sensitive radioimmunoassay (RIA) for measuring native porcine interferon gamma (nPoIFNγ).
- To characterize the nPoIFNγ produced by porcine lymphocytes stimulated with phorbol-myristate-acetate (PMA) and phytohemagglutinin (PHA).
- To assess the utility of the developed RIA as a complement to existing bioassays.
Main Methods:
- Development of a monospecific RIA using rabbit antibodies against recombinant porcine IFNγ (rPoIFNγ) immobilized on Sepharose.
- Stimulation of porcine lymphocytes with PMA and PHA, followed by RIA measurement of secreted nPoIFNγ.
- Fractionation of culture fluids using Concanavalin A Sepharose (Con A/Seph) and anti-rPoIFNγ-Seph to purify and characterize nPoIFNγ.
- Analysis of purified nPoIFNγ using PAGE-SDS electrophoresis and Western blotting.
Main Results:
- The developed RIA specifically detected nPoIFNγ, showing at least a two-fold increase in levels from stimulated lymphocytes compared to controls.
- nPoIFNγ was retained on Con A/Seph, indicating the presence of sugar residues, and was specifically adsorbed and eluted from the anti-rPoIFNγ-Seph matrix.
- Purified nPoIFNγ showed specific binding to the antibody and similar migration patterns to rPoIFNγ in electrophoresis.
- The RIA demonstrated high sensitivity and specificity, with eluted nPoIFNγ exhibiting significantly higher reactivity.
Conclusions:
- A novel and sensitive RIA for quantifying porcine interferon gamma has been successfully developed.
- The RIA allows for the accurate measurement of nPoIFNγ levels in biological samples.
- This immunoassay serves as a valuable tool for immunological research in pigs, complementing traditional bioassays.
Abstract:
Gamma interferon produced by porcine lymphocytes (nPoIFN gamma) in response to stimulation with phorbol-myristate-acetate (PMA) and phytohemagglutinin (PHA) was monitored by a radioimmunoassay (RIA). The RIA was developed with antibodies raised in rabbits against recombinant porcine IFN gamma (rPoIFN gamma) and made monospecific on a rPoIFN gamma/Sepharose matrix. This anti-PoIFN gamma antibody was shown to bind and neutralize rPoIFN gamma specifically and to cross-react with bovine IFN gamma but not with murine IFN gamma or porcine IFN alpha. The nPoIFN gamma levels produced by lymphocytes in response to PMA and PHA were at least two-fold higher than control lymphocytes as measured by the RIA in culture fluids. These culture fluids were fractionated on Concanavalin A Sepharose (Con A/Seph) and anti-rPoIFN gamma/Seph in attempts to evaluate the induced nPoIFN gamma further. The separation was monitored by RIA and showed that nPoIFN gamma was retained on Con A/Seph suggesting the presence of sugar residues on the molecules. Pools of Con A/Seph fractions, positive in RIA, were separated further on the anti-rPoIFN gamma/Seph matrix where a total adsorption of nPoIFN gamma occurred. On a weight basis, the eluates from the anti-rPoIFN gamma/Seph had a reactivity in RIA at least four times higher than the fractions derived from Con A/Seph. This indicated that the nPoIFN gamma remained immunochemically reactive after being eluted from Con A/Seph and that the separation on anti-rPoIFN gamma/Seph chromatography was specific. Purified nPoIFN gamma exhibited a major band with the same migration characteristics of rPoIFN gamma in PAGE-SDS electrophoresis and several minor bands when reacted with 125I anti-rPoIFN gamma antibody in Western blots. A total loss of reactivity to antibody after radioiodination of nPoIFN gamma, however, prevented the confirmation of these results by immunoprecipitation. A direct, rapid and sensitive immunoassay for measurement of the relative levels of nPoIFN gamma present in biological fluids is presented and this method could be a useful complement to bioassays for IFN gamma.
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