A stream cytometry-adapted fluorescent antibody to membrane antigen (FAMA) assay to detect IgG antibodies against varicella-zoster computer virus (VZV) was developed and tested in 62 serum samples, showing 90. latex agglutination assay have limited availability (5, 7, 9, 12, 14) Abacavir sulfate and may yield false-positive (2) and false-negative (10) results. Individuals with positive FAMA titers have a less than 3% risk of developing varicella after household exposure, while individuals with bad FAMA titers have a 75% risk (12). The disadvantages of the FAMA assay are its nonautomation, subjective interpretation, limited level, lengthy execution, and need for specific teaching (1, 6, 8, 11). There remains a need for a practical and reproducible assay that can determine susceptibility to varicella and confirm seroconversion following vaccination and exposure. FAMA test is an immunofluorescence assay that uses unfixed varicella-zoster computer virus (VZV)-infected human being embryonic lung fibroblast (HELF) cells incubated with serial 2-fold dilutions of sera. The cells Abacavir sulfate are then washed, incubated, and examined using fluorescence microscopy (16). We developed a circulation cytometry-adapted FAMA assay (flow-FAMA) that uses the same HELF cells (in our case, infected less than 48 h, having a cytopathic effect of less than 90%). Similarly to the standard FAMA assay, we incubated the infected cells in 25 l of diluted sera for 30 min, washed the cells in phosphate-buffered saline (PBS), and incubated them for 30 min in 25 l of diluted fluorescein-conjugated anti-human immunoglobulin G. After this second incubation and a second wash, the flow-FAMA assay process diverged from the original assay process. Rather than preparing the cells on a slide to be examined under a microscope, we resuspended the cells in 300 Abacavir sulfate l of calcium/magnesium-negative PBS and transferred them to circulation cytometry tubes. The labeled cells were then analyzed using a FACSCalibur (BD Sciences, San Jose, CA) and BD CellQuest software (Becton Dickinson, Franklin Lakes, NJ). We arranged a threshold for ahead scatter (FSC) at 760 and one for part scatter (SSC) at 550, and we collected 5,000 events for each sample. With the intention of assigning a quantitative value that corresponded with humoral immunity, we used FlowJo software 8.8.4 for Mac pc (Tree Star Inc., Ashland, OR) to produce two gates (both fixed throughout the experiments), one that excluded noise and a second that captured events representing humoral immunity (Fig. 1). By calculating the percentage of Abacavir sulfate events appearing within the borders of this second gate, we generated Abacavir sulfate a percent positivity for each sample. Fig. 1. FAMA positive-control (A) and negative-control (B) gatings using ahead scatter (FSC) and fluorescein isothiocyanate (FITC) axes as themes for analysis. Results are indicated as percentages of positivity inside oval gates (in the right-hand panels). … To judge flow-FAMA, we tested two band of samples blindly. Group I (= 20) (examples A to T in Desk 1) had been archived sera that were collected and kept since 1974. Of the 20 samples, 10 Mouse monoclonal to CD31.COB31 monoclonal reacts with human CD31, a 130-140kD glycoprotein, which is also known as platelet endothelial cell adhesion molecule-1 (PECAM-1). The CD31 antigen is expressed on platelets and endothelial cells at high levels, as well as on T-lymphocyte subsets, monocytes, and granulocytes. The CD31 molecule has also been found in metastatic colon carcinoma. CD31 (PECAM-1) is an adhesion receptor with signaling function that is implicated in vascular wound healing, angiogenesis and transendothelial migration of leukocyte inflammatory responses.
This clone is cross reactive with non-human primate.
were FAMA positive, obtained from subjects with a positive history of the disease and no disease after subsequent exposure, and 10 were FAMA negative, obtained from subjects with no history of varicella or vaccination, who later developed the clinical disease and FAMA seroconversion. To evaluate inter- and intra-assay variation, we tested each sample 5 or 6 times over a period of 4 days: once in triplicate and two or three times individually. Table 1. Results obtained with standard FAMA and flow cytometry-adapted FAMA assays Group II (= 39) (samples 1 to 39 in Table 1) were samples that tested as either negative or equivocal using the commercially available ELISA in our hospital. We chose these because they were problematic and typically sent to be tested by FAMA. The samples were tested in triplicate (on a single day) and the results averaged (Table 1). The positive control was a high-titer FAMA-positive serum sample obtained from a.