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1994-09-05
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Document 0837
DOCN M9480837
TI Immunomagnetic capture of CD4+ cells from whole blood for HIV detection
by PCR.
DT 9410
AU Piccoli SP; Ekeze TE; Gross S; Mehta RR; Kerschner JH; Immunicon Corp.,
Huntingdon Valley, PA.
SO Abstr Gen Meet Am Soc Microbiol. 1994;94:555 (abstract no. C-366).
Unique Identifier : AIDSLINE ASM94/94313108
AB Rapid diagnosis of HIV infection may be greatly facilitated by direct
identification of the provirus, regardless of the time course of
antibody response. Toward this end, we have developed a system to permit
specific isolation of CD4+ T-cells for subsequent genetic analysis by
PCR. Samples of whole blood (50-75% v/v in PBS) were incubated with an
anti-CD4 monoclonal antibody. A ferrofluid (colloidal magnetite, Fe3O4),
coated with goat anti-mouse Fc antibodies, was added to magnetically
label the target CD4+ cells. Samples were then subjected to a brief high
gradient magnetic separation (HGMS) to isolate the cells. To ensure
complete removal of erythrocytes, the cells were resuspended and
magnetically washed. Lysis was accomplished in a Proteinase K/Tween 20
buffer at ambient temperature. Following heat inactivation of the
enzyme, aliquots were subjected to PCR. All results were confirmed by
probe hybridization to the product. In an initial study, twelve blinded
samples from previously diagnosed patients or negative controls were
examined for the presence of HIV using this procedure. All samples were
found to be in agreement with serology and clinical diagnoses (10 HIV
seropositive and 2 HIV seronegative) by appropriate detection of the
presence or absence of the provirus. The sensitivity of this method was
found to be equivalent to others such as Ficoll-Hypaque cell separation
or erythrocyte removal by ammonium chloride lysis, followed by WBC lysis
to release DNA and/or phenol/chloroform extraction. Similar results with
mock-infected samples indicated a sensitivity limit of 10 HIV genome
equivalents added to the final cell lysate. CD4+ cells were isolated in
approximately 95% yield with approximately 95% purity as analyzed by
flow cytometry. These data suggest that biologically active ferrofluids
represent a rapid (< 30 minutes) and efficient method for preparation of
peripheral blood cell subsets for diagnostic analysis by PCR.
DE Acquired Immunodeficiency Syndrome/*DIAGNOSIS Cell Separation/*METHODS
Flow Cytometry Human HIV/*ISOLATION & PURIF *HIV Seronegativity HIV
Seropositivity/*DIAGNOSIS Lymphocyte Subsets/CYTOLOGY/PATHOLOGY
Magnetics Polymerase Chain Reaction/*METHODS Proviruses/ISOLATION &
PURIF T4 Lymphocytes/CYTOLOGY/*MICROBIOLOGY/PATHOLOGY MEETING ABSTRACT
SOURCE: National Library of Medicine. NOTICE: This material may be
protected by Copyright Law (Title 17, U.S.Code).