Quantitation of proteins and internal antigen pools by a monoclonal sandwich radioimmune assay

We describe a monoclonal sandwich radioimmune assay (MSRA) that is capable of measuring 0.5 fmoles of protein antigens. The basis of the assay is the ability of most rabbit heteroantisera directed against protein antigens to precipitate labeled monoclonal Fab-antigen complexes. The assay offers the advantages of specificity, sensitivity, linearity and rapidity. The MSRA circumvents the need for preparation of either highly purified protein antigens for use in radioimmune inhibition assays or affinity purified antisera for sandwich immunoassays. Utilizing variations of MSRA protocols, we determined that 48% of FcR gamma 2b/gamma 1, 34% of an 90,000 Mr glycoprotein identified by monoclonal antibody 2D2C, and 24% of an 82,000 Mr glycoprotein identified by monoclonal antibody 2E2A were accessible to labeled Fab probes at the plasma membrane. The total amount of antigen per cell was determined by dividing the amount of antigen at the cell surface (determined from binding saturation assays) by the percentage of surface/total antigen. This value of total antigen agrees well with the total cellular antigen pool determined by MSRA after appropriate corrections for probe saturation and recovery of pre-formed Fab-antigen complexes were made.