12 research outputs found
An evaluation of commercial fluorescent bead-based luminex cytokine assays.
The recent introduction of fluorescent bead-based technology, allowing the measurement of multiples analytes in a single 25-50 microl 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-plex kit; LINCO Inc's 29-plex kit; and RnD System's Fluorokine-Multi Analyte Profiling (MAP) base kit A and B). The LINCO 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-gamma measured in antigen stimulated whole blood culture supernatants by the LINCO Inc 29-plex, RnD Fluorokine-(MAP) and RnD system IFN-gamma 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
Identification of immune correlates of natural protection against tuberculosis in a population with a high incidence of latent infection
Thesis (MScMed)--Stellenbosch University, 2008.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.AFRIKAANSE OPSOMMING: Studie-area Hierdie studie was gedoen in die Tygerberg area van Kaapstad in Suid-Afrika. Agtergrond ‘n Derde van die wêreld se bevolking is latent geïnfekteer met Mycobacterium tuberculosis en korrelate van beskerming teen die siekte moet geïdentifiseer word om die ontwikkeling van ‘n effektiewe enstof te fasiliteer. Die produksie van IFN-γ is welbekend as ‘n immuunkorrelaat van beskerming teen tuberkulose (TB), maar ander immuunreguleerders speel ook ‘n belangrike rol in beskermende immuniteit. Die doelwitte van hierdie projek was drievoudig: (i) om belowende TB-entstof kandidate te identifiseer deur die sifting van ‘n paneel van nuwe MTB antigene mbv die in vitro stimulasie van volbloed kulture, ii) om ander sitokiene en chemokiene as immuunkorrelate van beskerming te identifiseer deur van die Luminex fluorescent bead-based tegniek gebruik te maak, en (iii) om die twee tegnieke te vergelyk op grond van hul prestasie as prognostiese of siftings metodes in latente infeksie. Metodes Antigeen siftings studie Volbloed van 57 volwasse en adolessente deelnemers, geïdentifiseer as latent geïnfekteerde individue, was gestimuleer met ‘n paneel van 78 nuwe TB-spesifieke DosR- or R-gekodeerde antigene. Die 7-dae kultuur supernatante was gebruik in ‘n IFN-γ ELISA om die hoeveelheid IFN-γ produksie the kwantifiseer. Luminex assay studie Volbloed kultuur supernatante van 15 HIV negatiewe, TST positiewe volwassenes was gebruik in die Luminex LINCO 21-plex cytokine assay. Dit was gedoen om die tipes en hoeveelheid ander LTBI-geassosieerde sitokienes te identifiseer wat geproduseer word na stimulasie met 9 TB-spesifieke rekombinante antigene. Resultate In die antigeen siftings studie is gevind dat die meerderheid van die 78 getoetste proteïene ‘n positiewe IFN-γ reaksie kon induseer. Vir die kontroles was die frekwensie van reaksies die hoogste na stimulasie met ESAT-6 en TesatCFP-10 (80 – 85% van reageerders). Tien latensie antigene was gereeld herken deur 19 – 45% van deelnemers en vyf reaktiverings-antigene het ‘n positiewe reaksie in 15 – 48% van reageerders gestimuleer. Die kategorie van antigene wat die meeste en hoogste response veroorsaak het, was die resusitasie-promoterende faktors (Rpf). Meer as 30% van deelnemers het op al 5 Rpfs gereageer en die vlak van reaksies was gelyk verdeel in die lae en matig-tot-hoog vlakke, met ‘n addisionele 5% van reaksies in die hoë (>1000pg/ml) reeks. In die Luminex studie het die positiewe stimulant TesatCFP-10 konsekwent die positiewe uitdrukking van die meeste sitokiene geïnduseer. Saam met dit het die latente antigene Rv1733c, Rv0569 en Rv2029c ook matige-toe-hoë vlakke van sitokien uitdrukking geïnduseer. ‘n Th1-gebaseerde sitokien profiel was waargeneem, met die begunstigde uitdrukking van pro-inflammatoriese en sel-gemedieerde sitokiene soos IFN-γ, TNF-α, IP-10, MIP1-α en G-CSF. Th2 sitokiene IL-4, IL-5, IL- 13 en eotaksien was of baie sleg uitgedruk of onder naspeurbare vlakke uitgedruk. ‘n Baie sterk induksie van IL-6, IL-8 en MCP-1 was waargeneem, maar hierdie
sitokiene/chemokiene assosiasie stel moontlik kontaminasie van die rekombinante
antigene met bakteriële endotoksiene voor.
Samevatting
Die reaksiepatroon wat in hierdie studie tussen die twee toetse waargeneem is, was
grootliks ‘n Th1-gebaseerde uitdrukkingsprofiel vir latente infeksie met TB. Die
volbloed ELISA metode is a betroubare gevestigde toets vir die kwantifisering van
IFN-γ in kultuur supernatante, wat ook in hierdie studie bewys is om effektief te wees.
Hierdie studie het gedemonstreer dat die nuwe TB-spesifieke antigene effektief
positiewe IFN-γ response in mense met LTBI induseer. Die multipleks tegnologie is
ook ‘n baie veelsydige en sensitiewe toets, wat in staat is om veelvoudige analite
gelyktydig in een monster te kan opspoor. In hierdie studie was dit veral waardevol
in die identifisering van ander moontlike antigene as prognostiese kandidate en
sitokiene as immuunbemiddelaars in TB-infeksie
Recoveries of the Bio-Rad 17-plex assay.
<p>Un-stimulated whole blood culture supernatant samples from healthy donors were spiked at three (experiment 1) and five (experiment 2) different concentrations (2–8062 pg/ml) with the standard from the Bio-Rad 17-plex kit. Samples were assayed in duplicate and read at high and low RP1 targets on the Bio-plex system instrument. Recoveries were calculated for each of the cytokines in the panels for each of the spiked concentrations. The figure shows the recoveries obtained for each individual cytokine after two independent experiments.</p
Detailed statistics of Bio-Rad 17-plex assay recoveries (two independent experiments).
<p>Unstimulated whole blood culture supernatant samples were spiked at different concentrations with recombinant cytokines. Samples were analysed on the Bio-plex system instrument and the recovery of each cytokine in assessed after subtraction of the endogenous levels of cytokines.</p
Recoveries of the Linco 29-plex assay.
<p>Un-stimulated whole blood culture supernatant samples from healthy donors were spiked at five (experiment 1) and two (experiment 2) different concentrations ranging from 10–5000 pg/ml with the standards from the LINCO 29-plex kit. Samples were assayed in duplicate and read at high and low RP1 targets on the Bio-plex system instrument. Recoveries were calculated for each of the cytokines in the panels for each of the spiked concentrations. The figure shows the recoveries obtained for each individual cytokine after two independent experiments.</p
Detailed statistics of Linco-plex assay recoveries (two independent experiments).
<p><b>(−)</b> Cytokines not included in the second test.</p><p>Unstimulated whole blood culture supernatant samples were spiked at different concentrations with recombinant cytokines. Samples were analysed on the Bio-plex system instrument and the recovery of each cytokine assessed after subtraction of the endogenous levels of cytokines.</p
RnD System's Fluorokine-MAP kits assay recoveries detailed statistics (two independent experiments).
<p>(*) Cytokines included in both experiment 1 and 2.</p><p>Unstimulated whole blood culture supernatant and serum samples were spiked at different concentrations with recombinant cytokines. After samples were measured and the level endogenous levels of cytokines subtracted the recovery of each cytokine was assessed. Only IFN-γ, ΤΝF-α and IL-4 were included in the second experiment.</p
Bio-Rad human 17-plex expected and observed cytokine concentrations and recovery (experiment 1).
<p>Unstimulated whole blood culture supernatant samples were spiked at three different concentrations of recombinant cytokines. Samples were analysed on the Bio-plex system instrument and the recovery of each cytokine in the panel assessed. The expected concentrations, after subtraction of the endogenous levels of cytokines, are represented.</p
Linco 29-plex assay reproducibility.
<p>Five aliquots of the same PHA-stimulated whole blood supernatant were produced and each aliquot was run on different plates and on different days.</p
Correlation between ELISA, LINCO 29-plex, Bio-Rad 17-plex and RnD Systems Fluorokine-MAP 13-plex assays.
<p>10 Whole blood supernatant from a healthy donor were stimulated with antigens in six day assays. The table shows the intra-class correlation coefficients (ICC) of agreement and consistency as well as Pearson correlation coefficients (r) between measurements obtained with the different luminex kits and the ELISA.</p