Comparison of different immunostaining techniques and monoclonal antibodies to the lower matrix phosphoprotein (pp65) for optimal quantitation of human cytomegalovirus antigenemia

Abstract

The main parameters of immunostaining techniques, i.e., the type of fixative, immunocytochemical reaction, and quality of monoclonal antibodies (MAbs), for quantitation of human cytomegalovirus (HCMV) antigenemia in peripheral blood polymorphonuclear leukocytes (currently performed by the indirect immunofluorescence or immunoperoxidase reaction by using MAbs to HCMV pp65) were investigated in order to optimize procedural steps and reagents. Significantly better results (in terms of the number of positive cells) were obtained on multiple cytospin preparations from heart transplant recipients with HCMV viremia when we used (i) formalin instead of methanol-acetone fixation and (ii) the indirect immunofluorescence reaction instead of the immunoperoxidase reaction, the avidin-biotin complex method, or the alkaline phosphatase antialkaline phosphatase procedure. In addition, comparison of the staining capabilities of three MAbs to pp65, which were developed in the laboratory and which were reactive to different epitopes of the protein, with a commercially available MAb (Clonab CMV) for determination of HCMV antigenemia showed that, while individual MAbs did not provide better results, the pool of MAbs detected a significantly higher number of positive peripheral blood polymorphonuclear leukocytes than Clonab CMV did. In addition, the sensitivity of the pool in detecting patients with low levels of viremia (less than 5/2 x 10(5) cells inoculated) as antigenemia positive was 100%, whereas the sensitivity of Clonab CMV was 47%. No differences in the specificities between the two MAb preparations were observed.