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

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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 (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.

Original languageEnglish
Pages (from-to)1232-1237
Number of pages6
JournalJournal of Clinical Microbiology
Volume30
Issue number5
Publication statusPublished - 1992

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Phosphoproteins
Cytomegalovirus
Monoclonal Antibodies
Viremia
Indirect Fluorescent Antibody Technique
Neutrophils
Fixatives
Avidin
Biotin
Acetone
Phosphoric Monoester Hydrolases
Formaldehyde
Alkaline Phosphatase
Methanol
Epitopes
Cell Count
Staining and Labeling
Proteins

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

Cite this

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title = "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 (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.",
author = "G. Gerna and Revello, {M. G.} and E. Percivalle and F. Morini",
year = "1992",
language = "English",
volume = "30",
pages = "1232--1237",
journal = "Journal of Clinical Microbiology",
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T1 - Comparison of different immunostaining techniques and monoclonal antibodies to the lower matrix phosphoprotein (pp65) for optimal quantitation of human cytomegalovirus antigenemia

AU - Gerna, G.

AU - Revello, M. G.

AU - Percivalle, E.

AU - Morini, F.

PY - 1992

Y1 - 1992

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