Flow Cytometric Indirect Immunofluorescence Assay with High Sensitivity and Specificity for Detection of Antibodies to Human Immunodeficiency Virus (HIV)

Sligh, Julie M.; Roodman, Stanford T.; Tsai, Chun-Chin

doi:10.1093/ajcp/91.2.210

Cited by 15 publications

(12 citation statements)

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“…Using particles of different sizes coated with p31, gp120, p24, and gp41 antigens from HIV-1, Scillian et al (290) were able to detect and quantify the specific antibodies. An FCM immunofluorescence assay (FIFA) with high sensitivity and specificity was developed by Sligh et al (294) to detect antibodies to HIV-1 by using HIV-1-infected cell lines. The cells are incubated with the sera to be tested, and incubation with an FITCconjugated anti-human Ig and FCM allows the quantification of HIV-1 antibodies in the sera.…”

Section: Serological Diagnosismentioning

confidence: 99%

Applications of Flow Cytometry to Clinical Microbiology

Alvarez-Barrientos¹,

Arroyo²,

Cantón³

et al. 2000

Clinical Microbiology Reviews

161

112

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Section: Serological Diagnosismentioning

confidence: 99%

Applications of Flow Cytometry to Clinical Microbiology

Alvarez-Barrientos¹,

Arroyo²,

Cantón³

et al. 2000

Clinical Microbiology Reviews

161

112

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“…In addition, several variations of Enzymelinked immunosorbent assay (ELISA) have also been proposed by several authors as a possible alternative to cell-culture tests (ATANASIU et al, 1977;ELMGREN & WANDELER, 1996;ESTERHUYSEN et al, 1995;NICHOLSON & PRESTAGE, 1982;PERRIN et al, 1986;PIZA et al, 1999). Flow Cytometry, a methodological analysis approach based upon refracted light, has been used for detecting antibodies to different viruses such as HCMV (McHUGH et al, 1986 and1988), HSV (SCILLIAN et al, 1989) and HIV (SLIGH et al, 1989). We have recently reported the use of Flow Cytometer for rabies virus detection (BORDIGNON et al, 2002) and the present study describes the standardization of a Flow Cytometry technique for detection and quantification of rabies VNA.…”

Section: Introductionmentioning

confidence: 99%

Calculating rabies virus neutralizing antibodies titres by flow cytometry

Bordignon

Comin

Ferreira³

et al. 2002

Rev. Inst. Med. trop. S. Paulo

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The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton Dickinson FACSCalibur flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity.

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“…We have developed the recombinant flow cytometric indirect immunofluorescence assay (r-FIFA), which is based on the flow cytometric indirect immunofluorescence assay (FIFA) (24). The purified insoluble forms of the HIV-1 gag precursor, Gag-gp41 chimeric proteins, the pol precursor polyprotein Pol97, and cell-associated gp160 expressed in insect cells by baculovirus vectors instead of HIV-1-infected human cells (H9) were used directly as carriers and antigens for r-FIFA.…”

mentioning

confidence: 99%

“…Pol97 is an excellent antigen for use in the detection of antibodies to HIV-1 and HIV-2 (20). Furthermore, the use of insoluble forms of HIV-1 recombinant protein for r-FIFA would solve the problems of antibody cross-reactivity to human cell antigens and the biohazard concerns of the original FIFA (24). In this report, we compare the sensitivity and specificity of r-FIFA with those of the licensed screening and confirmatory tests using HIV-1 seroconversion panels from Boston Biomedica Inc. (BBI) and the HIV-1 seroconversion specimens provided by the Provincial Public Health Laboratory of Ontario, Toronto, Ontario, Canada (PHL).…”

mentioning

confidence: 99%

“…A mixture of Gag-p45 and Gag-gp41-C was used as the antigen for testing the samples.VOL. 34, 1996HIV-1 ANTIBODY DETECTION 1415 on July 10, 2020 by guest http://jcm.asm.org/ and adding 3 standard deviations (SDs) as described previously(24). For example, the cutoff value of fluorescence intensity for Gag-p45 was calculated as follows: for IgG FITC, x S/N ϭ 0.960, 1 SD ϭ 0.266, and cutoff ϭ x S/N ϩ 3 SDs ϭ 1.75; for IgM R-PE, x S/N ϭ 1.666, 1 SD ϭ 0.581, and cutoff ϭ x S/N ϩ 3 SDs ϭ 3.41; and for IgA R-PE, x S/N ϭ 1.270, 1 SD ϭ 0.604, and cutoff ϭ x S/N ϩ 3 SDs ϭ 3.08.…”

mentioning

confidence: 99%

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Flow cytometric immunofluorescence assay for detection of antibodies to human immunodeficiency virus type 1 using insoluble precursor forms of recombinant polyproteins as carriers and antigens

Birch

Balaskas

et al. 1996

J Clin Microbiol

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A new serological assay, the recombinant flow cytometric immunofluorescence assay (r-FIFA), was developed for the early detection of human immunodeficiency virus type 1 (HIV-1) antibodies by using recombinant insoluble forms of HIV-1 Gag-p45, Gag-gp41 chimeric protein, gp160, and Pol97 polyprotein as antigens and autologous carriers through flow cytometry. These recombinant proteins were expressed in insect cells by a baculovirus expression system. Eight anti-HIV-1 seroconversion panels, a low-titer anti-HIV-1 panel from Boston Biomedica Inc. (BBI), and three HIV-1 seroconversion specimens from the Provincial Health Laboratory of Ontario, Toronto, Ontario, Canada (PHL), were tested and analyzed by r-FIFA. In sensitivity comparisons between r-FIFA and tests licensed by the U.S. Food and Drug Administration, which were used to test all of the HIV-1 panels from BBI, detection of HIV-1 antibody by r-FIFA was on average greater than 20 days earlier than that by enzyme immunoassay. The sensitivity of r-FIFA has permitted the detection of HIV-1specific immunoglobulin G (IgG), IgM, and IgA antibodies during seroconversion. A kinetic analysis of HIV-1 antibody production by r-FIFA has shown that either IgG or IgM, or both, can be detected, depending on the phase and type of the immune response in the HIV-1-infected individual. Both primary and secondary immune responses were observed during this period. The r-FIFA results suggest that implementation of r-FIFA may significantly reduce the ''window'' period from the time of infection to the time of seroconversion, with earlier detection of antibodies after initial infection. This would also make it possible for us to understand the immune response and the precise mechanisms of immunopathogenesis in the early period of HIV-1 infection.

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Flow Cytometric Indirect Immunofluorescence Assay with High Sensitivity and Specificity for Detection of Antibodies to Human Immunodeficiency Virus (HIV)

Cited by 15 publications

References 0 publications

Applications of Flow Cytometry to Clinical Microbiology

Applications of Flow Cytometry to Clinical Microbiology

Calculating rabies virus neutralizing antibodies titres by flow cytometry

Flow cytometric immunofluorescence assay for detection of antibodies to human immunodeficiency virus type 1 using insoluble precursor forms of recombinant polyproteins as carriers and antigens

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