A simple, rapid, and sensitive scintillation proximity assay for the determination of levels of guinea-pig interleukin-5 in bronchoalveolar lavage samples.
This article describes the development and validation of a scintillation proximity assay (SPA) sensitive for guinea-pig interleukin-5 (IL-5). SPA beads were coated with TRFK-5, a monoclonal antibody directed against mouse IL-5, which is known also to bind guinea-pig IL-5. The assay is a simple competitive binding assay between [125I]-rh-IL-5 and the IL-5, in a sample of guinea-pig bronchoalveolar lavage fluid (BALF), for the binding site on the TRFK-5-coated beads. IL-5 levels in BALF ([IL-5]BALF) were shown to increase in guinea-pigs sensitized to ovalbumin (OvA) and challenged with an OvA inhalation. This occurred at a time (24 h) after challenge when there was also a marked eosinophilia. The assay was validated by treating guinea-pigs with a second antibody, Genzyme 2374-01, directed against IL-5. Treatment with this antibody resulted in a significant reduction of the antigen-induced eosinophilia and concentration of [IL-5]BALF. This observation confirms that the IL-5 identified in BALF also cross-reacts with the antibody Genzyme 2374-01. Interestingly, plasma from sensitized, but unchallenged, guinea-pigs also contained detectable levels of IL-5, and the stimulation of plasma protein extravasation (PPE) within the airways with inhaled histamine also induced a rise in [IL-5]BALF. These observations suggest that the plasma may be an additional source of the IL-5 present in the airways of antigen-challenged guinea-pigs.
Danahay
H
H
Pharmacology Department, Welsh School of Pharmacy, University of Wales Cardiff, Cathays Park, U.K.
Broadley
K J
KJ
McCabe
P J
PJ
Nials
A T
AT
Sanjar
S
S
D000280:Administration, Inhalation; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D000917:Antibody Formation; D001667:Binding, Competitive; D001798:Blood Proteins / Q000378:metabolism; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry* / Q000166:cytology; D004802:Eosinophilia / Q000139:chemically induced / Q000188:drug therapy; D006168:Guinea Pigs; D006632:Histamine / Q000008:administration & dosage / Q000276:immunology / Q000633:toxicity; D015848:Interleukin-5 / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008863:Microspheres; D010047:Ovalbumin / Q000008:administration & dosage / Q000276:immunology / Q000633:toxicity; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012588:Scintillation Counting
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D015848:Interleukin-5; D011994:Recombinant Proteins; D006632:Histamine; D010047:Ovalbumin