Radioimmunoassay for murine lactoferrin, a protein marker of myeloid and mammary epithelial secretory cell differentiation.

A radioimmunoassay was developed for murine lactoferrin (LF), a non-heme, iron-binding glycoprotein which appears to be a specific biochemical marker of differentiation in several cell types. Lactoferrin was labeled with 125I by the chloramine-T method to yield a product having 20 muCi/mug protein and an isotope incorporation of 0.6 atoms of 125I per molecule. Separation of bound and free lactoferrin was accomplished by either of two procedures, a double-antibody technique or precipitation in the presence of 50% saturated ammonium sulfate. The entire assay, including counting, was accomplished in less than 2 days and had a lower limit of sensitivity and a range of 1 ng/ml and 1-32 ng/ml, respectively, using rabbit antiserum in a dilution of about 1:10,000. The binding between LF and rabbit antiserum exhibited two association constants having values of 1.8 x 10(11) and 1 x 10(9) l/mole. The assay was specific for lactoferrin and no cross-reactivity was observed with transferrin, a similar non-heme, iron-binding glycoprotein. Human lactoferrin specifically reacted with anti-mouse lactoferrin, but the binding was approximately 8000 times weaker than observed with mouse lactoferrin. Values for lactoferrin in milk and bone marrow granulocytes were obtained which agreed with levels obtained using other methods.