Browsing by Author "Golakai, Hawa Jande"

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    An evaluation of commercial fluorescent bead-based luminex cytokine assays
    (Public Library of Science, 2008-07-02) Siawaya, Joel Fleury Djoba; Roberts, Teri; Babb, Chantal; Black, Gillian; Golakai, Hawa Jande; Stanley, Kim; Bapela, Nchinya Bennedict; Hoal, Eileen; Parida, Shreemanta; Van Helden, Paul; Walzl, Gerhard
    The recent introduction of fluorescent bead-based technology, allowing the measurement of multiples analytes in a single 25-50 μl sample has revolutionized the study of cytokine responses. However, such multiplex approaches may compromise the ability of these assays to accurately measure actual cytokine levels. This study evaluates the performance of three commercially available multiplex cytokine fluorescent bead-based immunoassays (Bio-Rad's Cytokine 17-piex kit; LINCO Inc's 29-plex kit; and RnD System's Fluorokine-Multi Analyte Profiling (MAP) base kit A and B). The LINEO Inc kit was found to be the most sensitive assay for measuring concentrations of multiple recombinant cytokines in samples that had been spiked with serial dilutions of the standard provided by the manufacturer, followed respectively by the RnD Fluorokine-(MAP) and Bio-Rad 17-plex kits. A positive correlation was found in the levels of IFN-γ measured in antigen stimulated whole blood culture supernatants by the UNCO inc 29-plex, RnD Fluorokine-(MAP) and RnD system IFN-γ Quantikine ELISA kits across a panel of controls and stimulated samples. Researchers should take the limitation of such multiplexed assays into account when planning experiments and the most appropriate use for these tests may currently be as screening tools for the selection of promising markers for analysis by more sensitive techniques. © 2008 Djoba Siawaya et al.
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    Identification of immune correlates of natural protection against tuberculosis in a population with a high incidence of latent infection
    (Stellenbosch : Stellenbosch University, 2008-03) Golakai, Hawa Jande; Walzl, Gerhard; Black, Gillian; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
    ENGLISH ABSTRACT: Setting This study was conducted in the Tygerberg area of Cape Town in South Africa. Background A third of the world’s population is latently infected with Mycobacterium tuberculosis, and correlates of protection against progression to active disease urgently need to be identified to facilitate the development of an effective vaccine against the disease. The production of IFN-γ is recognised as an immune correlate of protection from tuberculosis, but other immune regulators have been implicated in playing a significant role in protective immunity. The aims of this project were three-fold: (i) to identify promising TB vaccine candidates by screening a panel of novel MTB antigens, by stimulating whole blood cultures in vitro with the novel proteins and quantifying the level of IFN-γ production, (ii) to identify other cytokines and chemokines that may be immune correlates of protection using the Luminex fluorescent bead-based technique and (iii) to compare the performance of the two techniques. Methods Antigen Screening study Whole blood of 57 adult and adolescent participants defined as latently infected individuals was stimulated with a panel of 78 novel TB-specific, DosR- or RD1-encoded antigens. The 7-day culture supernatants were used in IFN-γ ELISA to quantify the level of IFN-γ production. Luminex Assay study Whole blood culture supernatants of 15 HIV negative, TST positive adults were used in the Luminex LINCO 21-plex cytokine assay. This was done to determine which of 21 cytokines, that may be LTBI-associated cytokines, were produced after stimulation with 9 TB-specific recombinant antigens, and to quantify their level of expression. Results In the antigen screening study, it was found the majority of the 78 proteins tested were able to induce a positive IFN-γ response. The classic TB antigens were used as controls, and the frequency of responses was highest after stimulation with ESAT-6 and TesatCFP10 (80 – 85% of responders). Ten latency antigens elicited an IFN-γ response in 19 – 45% of participants, and five reactivation antigens stimulated a positive reaction in 15 – 48% of responders. The category of antigens that elicited the most frequent and highest responses overall was the resuscitation-promoting factors (Rpf). Over 30% of participants responded to all 5 Rpfs, and the level of responses were equally divided in the low and moderate-to-high levels, with an additional 5% of responses in the high (>1000pg/ml) range. In the Luminex study, the positive stimulant TesatCFP10 consistently induced expression of most cytokines. In addition latency antigens Rv1733c, Rv0569 and Rv2029c also induced moderate-to-high level cytokine expression. A Th1-biased cytokine profile was observed, with the preferential expression of pro-inflammatory and cell-mediated cytokines like IFN-γ, TNF-α, IP-10, MIP1-α and G-CSF being produced. Th2 cytokines IL-4, IL-5, IL-13 and eotaxin were very poorly expressed or were not expressed at detectable levels. A very strong induction of IL-6, IL-8 and MCP-1 was observed, but this cytokine/chemokine association suggested contamination of the recombinant antigens with bacterial endotoxins. Conclusion In this study of latently infected individuals, the pattern of response observed for both assays is largely a Th1-biased expression profile. The whole blood ELISA method is a well-established assay for quantifying IFN-γ in culture supernatants, and has proven to be effective here. This study has demonstrated, in humans with LTBI, immune recognition of these novel MTB-specific antigens as illustrated by the positive IFN-γ levels induced after stimulation. The multiplex technology is also a very versatile and sensitive assay, capable of detecting multiple analytes simultaneously in one sample. The multiplex has been valuable here in identifying some antigens as potential vaccine candidates, and a subset of cytokines as potential immune mediators and prognostic indicators in TB infection.