Quantitative immunofluorescent assay of full-length, recombinant CD4 in solution and mapping of its epitopes

A specific, rapid, and sensitive method for the detection of CD4 in solution was developed using pairs of fluorescently stained monoclonal antibodies which do not cross-compete. The assay is quantitated by flow cytometry using Simply Cellular microbeads (SC beads) as the primary support for the first anti-CD4 mAb. This method uses the standard conditions for anti-CD4 monoclonal antibody binding, washing, detection, and quantitation by flow cytometry of the CD4 antigen either bound to the SC beads or expressed on the cell surface. The monoclonal antibody used (Leu 3a PE) is the standard reference used to evaluate the CD4 concentration. This method differs from ELISA techniques, which need an antigen standard curve and thus can be influenced by the quality and source of the antigen. This type of assay is also a procedure which enables determination of the level of oligomerization of the bound antigen. It can be used for any antigen to which monoclonal antibodies recognizing at least two distinct epitopes are available. The use of soluble or full-length CD4 derivatives as potential therapeutic agents against AIDS, would benefit from a precise quantitation of the CD4 molecules which still have their proper tertiary structure.