Important note: Information in this article was accurate in 1994. The state of the art may have changed since the publication date.
Immunomagnetic capture of CD4+ cells from whole blood for HIV detection by PCR.
Abstr Gen Meet Am Soc Microbiol. 1994;94:555 (abstract no. C-366). Unique Identifier : AIDSLINE ASM94/94313108 Piccoli SP; Ekeze TE; Gross S; Mehta RR; Kerschner JH; Immunicon Corp., Huntingdon Valley, PA.
Abstract:
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.
Keywords: Acquired Immunodeficiency Syndrome/*DIAGNOSIS Cell Separation/*METHODS CD4-Positive T-Lymphocytes/CYTOLOGY/*MICROBIOLOGY/PATHOLOGY Flow Cytometry Human HIV/*ISOLATION & PURIF *HIV Seronegativity HIV Seropositivity/*DIAGNOSIS Lymphocyte Subsets/CYTOLOGY/PATHOLOGY Magnetics Polymerase Chain Reaction/*METHODS Proviruses/ISOLATION & PURIF ABSTRACT 941030
M94A0846
AEGiS presents published material, reprinted with permission and neither endorses nor opposes any material. All information contained on this website, including information relating to health conditions, products, and treatments, is for informational purposes only. It is often presented in summary or aggregate form. It is not meant to be a substitute for the advice provided by your own physician or other medical professionals. Always discuss treatment options with a doctor who specializes in treating HIV.
Important note: Information in this article was accurate in 1994. The state of the art may have changed since the publication date.
