A rapid screening technique for identifying murine monoclonal antibodies to human apolipoprotein A‐I isoforms by Western blot analysis

A rapid method for determining the reactivity of murine monoclonal antibodies with apolipoprotein A‐I (Apo A‐I) isoforms has been developed. The method consists of a rapid (1–2 h) prescreening of fusion well culture fluids by using an automated immunofluorescent assay (IF A) to identify fusion well cultures producing monoclonal antibody against Apo A‐I and high‐density lipoprotein (HDL). Immediately following the IFA procedure, immunodetection of the reactivity pattern of selected fusion well culture fluids is determined by using transblotted isoforms of Apo A‐I previously separated by isoelectric focusing. Strips are prepared and stored at 4 °C in protein containing blocking solution before use. Preselected culture fluids could be tested within 6 h for qualitative reactivity with Apo A‐I isoforms. Using this procedure we selected several monoclonal antibody‐producing cultures for further studies on HDL metabolism based on their differing reactivities with Apo A‐I isoforms. This technique should be easily transferable to other laboratories for assessing proteins having multiple isoforms since the crucial steps of separation and blotting can be optimized and performed before actual testing for antibody reactivity.