The CMV antigenemia assay was developed using a cocktail of two monoclonal antibodies (C10/C11) directed against CMV lower matrix protein pp65. The assay uses the C10/C11 cocktail in an indirect immunofluorescence staining of cytospin preparations of peripheral blood leukocytes. The first step in the CMV Brite™ Turbo method involves direct lysis of peripheral blood erythrocytes. Following lysis, the leukocytes are cytocentrifuged onto a slide, fixed and permeabilized to allow subsequent detection of CMV pp65 antigen. The presence of the CMV pp65 antigen is detected by the C10/C11 antibody cocktail and visualized by means of a specific secondary FITC-labeled antibody. CMVantigen-positive leukocytes exhibit homogeneous yellow-green polylobate nuclear staining when observed using a fluorescence microscope. The number of CMV antigen-positive cells is counted per duplicate stain. The whole procedure can be performed in approximately 2 hours.
Reagents for 110 tests including 5 control slides.
Reagent A – Erythrocyte Lysing Solution – Ammonium chloride solution containing <0.1% sodium azide. (10x). (Dilute before use in demineralized water). 200 ml
Reagent B – Fixative Solution – Formaldehyde, in PBS, containing < 0.1% sodium azide. (Dilute before use in PBS). (5x) 290 ml
Reagent C – Permeabilization Solution – Igepal Ca-630 and Newborn Calf Serum in PBS containing < 0.1% sodium azide.(5x) (Dilute before use in PBS). 290 ml
Reagent D – Monoclonal antibody C10/C11 (IgG1/IgG1) against CMV lower matrix protein pp65 containing < 0.1% sodium azide. (Ready to use) 4.0 ml
Reagent E – FITC-conjugated sheep anti-mouse Immunoglobulins – with Evans Blue containing < 0.1% sodium azide. (Ready to use) 4.0 ml
Control Slides – CMV Antigenemia Control Microscope Slides – Control slide
in a sealed pouch with desiccant. (Ready to use). 5 slides
Assay Time: ~ 2 hours
- Perform microscopic evaluation using an immunofluorescence microscope at 400 x
magnification. A higher magnification of 1000x may be used to increase resolution.
- Scan the whole surface of the spot. Two spots should be scanned per patient.
- Positive cells show homogenous yellow-green polylobate nuclear staining. Negative cells show no yellow-green nuclear staining.
Results and Interpretation:
- Results of evaluation of patient specimen slides are qualitative. The clinical significance of an increase or decrease in the number of antigenpositive cells during time in one patient has not been determined. Therefore, results should be
reported only as Positive or Negative, according to the following definitions:
- Positive – one or more CMV antigen- positive cells present per duplicate stain.
- Negative – no CMV antigen-positive cells present per duplicate stain.
- A minimum of approximately 50,000 specimen cells should be present in order to determine that a result is negative.