title: Amplified ELISPOT assay for the detection of HIV-specific antibody-secreting cells in subhuman primates.
abstract: A novel immunoenzyme amplification technique has been evaluated in an ELISPOT assay for the detection of antigen-specific antibody-secreting cells (ASC) in monkeys. In this assay, mononuclear cells containing putative ASC are incubated for a few hours in antigen-coated wells. Following removal of the cells, zones of solid phase bound antibodies secreted by individual ASC are visualized in four consecutive steps. First, a primary biotinylated anti-immunoglobulin (Ig) reagent is added followed by enzyme-labelled avidin. The amplification procedure comprises the addition of biotinylated anti-enzyme antibodies in the third stage, followed by enzyme-conjugated avidin and substrate. When evaluated in a modified ELISPOT assay for the detection of simian B cells secreting antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1), this amplification procedure proved to be suitable even when using anti-human Ig antisera as primary antibody reagents. This development should be useful for other ELISPOT assays where species specific anti-Ig reagents are not always available and, most importantly, for enumerating cells producing immunoreactive substances in such minute amounts that they may escape detection by conventional ELISPOT assays. Furthermore, a functional simian HIV-specific ELISPOT assay could prove valuable for assessing the humoral immunogenicity of future candidate vaccines against the acquired immunodeficiency syndrome (AIDS).
authors.affiliation: Department of Medical Microbiology and Immunology, University of Göteborg, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Eriksson
authors.forename: I
authors.initials: I
authors.lastname: Nordström
authors.forename: P
authors.initials: P
authors.lastname: Horal
authors.forename: S
authors.initials: S
authors.lastname: Jeansson
authors.forename: B
authors.initials: B
authors.lastname: Svennerholm
authors.forename: A
authors.initials: A
authors.lastname: Vahlne
authors.forename: J
authors.initials: J
authors.lastname: Holmgren
authors.forename: C
authors.initials: C
authors.lastname: Czerkinsky
mesh_terms: D000818:Animals; D000921:Antibody-Producing Cells / Q000276:immunology*; D015483:HIV Antibodies / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D007124:Immunoenzyme Techniques; D007132:Immunoglobulin Isotypes / Q000032:analysis; D008252:Macaca fascicularis / Q000276:immunology*
chemical_list: D015483:HIV Antibodies; D007132:Immunoglobulin Isotypes