Selective elution of immunoadsorbed anti-(human prolactin) immunoglobulins with enhanced immunochemical properties.
Specific anti-(human prolactin) immunoglobulin, isolated by immunoadsorption from a resolubilized Na2SO4 precipitate of a sheep antiserum, has been fractionated into antibody populations with a 10-fold range of affinity for antigen by using a new elution procedure. The procedure utilized acetonitrile (20%, v/v) in a gradient of pH, decreasing from pH 7 to pH2. The recovered immunoglobulin (74.3%) was eluted as a single peak after chromatography on Sephacryl S-300 and on radioiodination retained high immunoreactivity (91%). One antibody population, of low abundance, had an affinity constant (Ka = 8.6 x 10(10) l/mol) 5.7 times that of the unfractionated antiserum and, when used in radioimmunoassay standard curves, resulted in a 3.8-fold increase in the sensitivity of the assay. The bulk of adsorbed immunoglobulin was eluted at pH 4.3, thereby avoiding exposure to strong acid conditions and maintaining the integrity of the immunoglobulin molecule. In contrast, elution of immunoglobulin in the absence of acetonitrile could only be achieved at low pH (pH2) with minimal fractionation, and resulted in a poor recovery (37.1%) and loss of immunoreactivity (53%).
D000097:Acetonitriles; D000915:Antibody Affinity; D002850:Chromatography, Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007136:Immunoglobulins / Q000276:immunology / Q000302:isolation & purification*; D011388:Prolactin / Q000276:immunology*; D011863:Radioimmunoassay
D000097:Acetonitriles; D007136:Immunoglobulins; D011388:Prolactin