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

Differential Expression of Interleukin-4 (IL-4) and IL-4δ2 mRNA, but Not Transforming Growth Factor Beta (TGF-β), TGF-βRII, Foxp3, Gamma Interferon, T-bet, or GATA-3 mRNA, in Patients with Fast and Slow Responses to Antituberculosis Treatment▿

This study investigated interleukin-4 (IL-4), IL-4δ2, transforming growth factor beta (TGF-β), TGF-βRII, Foxp3, GATA-3, T-bet, and gamma interferon (IFN-γ) transcription in peripheral blood samples of adult pulmonary tuberculosis patients prior to and after 1 week of therapy. Twenty patients with positive results for sputum culture for Mycobacterium tuberculosis were enrolled and treated with directly observed short-course antituberculosis chemotherapy. Early treatment response was assessed. At the end of the intensive phase of treatment (month 2), 12 patients remained sputum culture positive (slow responders) and 8 converted to a negative culture (fast responders). Only the expression levels of IL-4 (4-fold decrease) and IL-4δ2 (32-fold increase) changed significantly during the first week of therapy in the 20 patients. No baseline differences were present between the responder groups, but fast responders had significantly higher IL-4 transcripts than slow responders at week 1. Fast responders showed a 19-fold upregulation and slow responders a 47-fold upregulation of IL-4δ2 at week 1. Only slow responders also showed a significant decrease in IL-4 expression at week 1. There were no significant differences in expression of TGF-β, TGF-βRII, Foxp3, IFN-γ, and GATA-3 between the groups. These data show that differential IL-4-related gene expression in the early stage of antituberculosis treatment accompanies differential treatment responses and may hold promise as a marker for treatment effect

Immune parameters as markers of tuberculosis extent of disease and early prediction of anti-tuberculosis chemotherapy response

This study investigates how the extent of pre-treatment radiological disease and early anti-tuberculous treatment response affect levels of selected circulating host immune markers. Twenty HIV-uninfected tuberculosis patients with BACTEC culture positivity for Mycobacterium tuberculosis at diagnosis and treated with directly observed short course anti-tuberculosis chemotherapy and 13 healthy community controls were enrolled. Serum samples were collected throughout treatment. After the intensive phase of treatment, 12\ua0patients remained sputum culture-positive (slow responders) and eight patients were culture negative (fast responders). C-reactive protein (CRP), soluble intercellular adhesion molecule-1 (sICAM-1), soluble urokinase plasminogen activator receptor (suPAR), soluble lymphocyte activation gene-3 (sLAG-3), granzyme B, soluble tumour necrosis factor receptor one and two (sTNFR I and sTNFR II) and soluble death receptor 5 (sDR5) concentrations were measured. High levels of CRP at diagnosis were found to be associated (p ≤ 0.05) with the presence of multiple cavities on chest x-rays and high levels of suPAR and sICAM-1 at diagnosis were associated (p ≤ 0.05) with the extent of alveolar disease. Also significant were the associations between the level of granzyme B (p ≤ 0.01) and LAG-3 (p ≤ 0.05) at diagnosis, and the size of the cavities. The combination of diagnosis and week one measurements of selected serological markers in mathematical models was able to identify the fast responders with up to 87.5% accuracy and the slow responders with up to 83.3% accuracy These preliminary results suggest that predictive models for differential early treatment responses using combinations of host markers hold promise

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

IFN-γ-based comparison of ELISA, LINCO 29-plex, Bio-Rad 17-plex and RnD Systems Fluorokine-MAP-13-plex assays.

<p>NA: Not applicable because the sample was not measured with the RnD Systems Fluorokine-MAP base kit A</p><p>Supernatant was generated using whole blood from a healthy donor (A-E) in six day assays stimulated with 5 differents Mtb derived antigens and with phytohaemagglutinin (PHA). The LINCO 29-plex measurement most closely resembled that of the RnD Systems ELISA in absolute value, and followed a similar trend, but absolute values for all three luminex assay fell below that of the ELISA.</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 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

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

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