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Development of quantitative suspension array assays for six immunoglobulin isotypes and subclasses to multiple Plasmodium falciparum antigens.

ABSTRACT: BACKGROUND:Quantitative suspension arrays are powerful immunoassays to measure antibodies against multiple antigens in large numbers of samples in a short time and using few microliters. To identify antigen targets of immunity for vaccine development against complex microbes like Plasmodium falciparum, such technology allows the characterization of the magnitude and antigenic specificity of Ig isotypes and subclasses that are important for functional responses. However, standardized assays are not widely available. METHODS:We developed six quantitative suspension array assays to measure IgG1, IgG2, IgG3, IgG4, IgM and IgE specific to multiple P. falciparum antigens. Secondary and tertiary antibodies, as well as human purified antibodies for standard curves, were tested among several commercially available sources. Positive and negative controls included plasmas from malaria hyper-immune African adults and from malaria-naïve European adults, respectively. Reagents were selected and optimal antibody and test sample dilutions established according to sensitivity, specificity and performance of the standard curves. The variability between replicates and plates was assessed with 30 test samples and controls. RESULTS:Assays were able to detect P. falciparum antigen-specific antibodies for all isotypes and subclasses in samples from malaria-exposed individuals, with low background signal in blank wells. Levels detected in malaria-naïve individuals were overall low except for IgM. For the IgG2 and IgE assays, a triple sandwich was required for sensitivity. Standard curves with 5-parameter logistic fit were successfully obtained in all assays. The coefficients of variation for measurements performed in different days were all <30%, and <5% when comparing duplicates from the same plate. CONCLUSION:The isotype/subclass assays developed here were sensitive, specific, reproducible and of adequate quantification dynamic range. They allow performing detailed immuno-profiling to large panels of P. falciparum antigens to address naturally- and vaccine-induced Ig responses and elucidate correlates of malaria protection, and could also be applied to other antigenic panels.

SUBMITTER: Vidal M

PROVIDER: S-EPMC5843563 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Development of quantitative suspension array assays for six immunoglobulin isotypes and subclasses to multiple Plasmodium falciparum antigens.

Vidal Marta M Aguilar Ruth R Campo Joseph J JJ Dobaño Carlota C

Journal of immunological methods 20180131

<h4>Background</h4>Quantitative suspension arrays are powerful immunoassays to measure antibodies against multiple antigens in large numbers of samples in a short time and using few microliters. To identify antigen targets of immunity for vaccine development against complex microbes like Plasmodium falciparum, such technology allows the characterization of the magnitude and antigenic specificity of Ig isotypes and subclasses that are important for functional responses. However, standardized assa ...[more]

PMID: 29397157

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Project description:BACKGROUND:Antibody responses to Plasmodium falciparum play a critical role in disease control. Finding reliable IgG biomarkers of protection is complicated by a parasite proteome of over 5000 proteins, some with polymorphisms. Studies of anti-malarial naturally acquired and vaccine immunity would benefit from a standard high-throughput immunoassay to measure multiple antibodies. A multiplex quantitative suspension assay to measure antigen-specific IgGs was developed and its precision (reproducibility and repeatability), dynamic range, limits of detection and quantification, and non-specific binding to different P. falciparum proteins tested. A set of 288 human plasma samples from a malaria-endemic region were analysed twice by two different operators. Another set of samples from 9 malaria-naïve and 10 malaria-exposed individuals were repetitively assayed during 22 consecutive days. Positive controls, negative controls, blanks and microspheres coated with bovine serum albumin were included in all assays. RESULTS:The multiplex quantitative suspension assay demonstrated low non-specific signal and good estimates of precision and reproducibility between operators. The overall mean of non-specific binding measured in 288 plasma samples was 32.83 to?±?44.81 median fluorescence intensity (MFI). Repeatability was 7.66%?±?15.89 between triplicates for all antigens and samples, being lower in samples from malaria-exposed than malaria-naïve individuals. No evidence of significantly different variance across days in MFI or arbitrary units (AU)/mL was found, assuming homogeneity of variance between days of analysis. Intra-class correlation coefficient between 22 days of analysis was 0.98 (0.97-0.98) for MFI units and 0.9 (0.87-0.93) for AU/mL. Reproducibility between operators for all samples and antigens had an overall adjusted correlation of 0.929 for MFI and 0.836 for AU/mL. CONCLUSIONS:This high-throughput multiplex immunoassay is simple and highly reproducible. This represents an asset for malaria vaccine studies involving CSP-specific antibodies and selected antigens for sero-epidemiological purposes. Measuring a multiplex antigen panel in a single reaction will help to assess not only vaccine immunogenicity but also potential malaria vaccine effects on naturally acquired immune responses. This will accelerate the identification of immune correlates of protection, down-selection of vaccine formulations, antigen discovery and guide second-generation vaccine design.

Project description:BackgroundMultiplex cytokine profiling systems are useful tools for investigating correlates of protective immunity. Several Luminex and flow cytometry methods are commercially available but there is limited information on the relative performance of different kits. A series of comparison experiments were carried out to determine the most appropriate method for our subsequent studies.MethodsTwo Luminex methods were compared, the Bio-Rad human 17-plex panel and the Invitrogen (formerly BioSource) human cytokine 10-plex kit, and two flow cytometry methods, the Becton Dickinson Human Th1/Th2 Cytokine Kit (CBA) and the Bender MedSystems Human Th1/Th2 11plex FlowCytomix Multiplex Kit. All kits were tested for the measurement of cytokines in supernatants collected from human leukocytes stimulated with viable Plasmodium falciparum infected red blood cells (iRBC) or P. falciparum schizont lysates.ResultsData indicated that the kits differed in sensitivity and reproducibility depending on the cytokine, and detected different quantities of some cytokines. The Bio-Rad 17-plex kit was able to detect more positive responses than the Invitrogen 10-plex kit. However, only when detecting IL-1, IL-6 or TNF did the two Luminex based methods correlate with one another. In this study, the flow cytometry based techniques were less variable and correlated better with one another. The two flow cytometry based kits showed significant correlation when detecting IFN-?, IL-2, TNF, IL-10 and IL-6, but overall the BD kit detected more positive responses than the Bender MedSystems kit.ConclusionsThe microsphere suspension array technologies tested differed in reproducibility and the absolute quantity of cytokine detected. Sample volume, the number of cytokines measured, and the time and cost of the assays also differed. These data provide an accurate assessment of the four techniques, which will allow individual researchers to select the tool most suited for their study population.

Project description:Reducing variability of quantitative suspension array assays is key for multi-center and large sero-epidemiological studies. To maximize precision and robustness of an in-house IgG multiplex assay, we analyzed the effect of several conditions on variability to find the best combination. The following assay conditions were studied through a fractional factorial design: antigen-bead coupling (stock vs. several), sample predilution (stock vs. daily), temperature of incubation of sample with antigen-bead (22°C vs. 37°C), plate washing (manual vs. automatic) and operator expertise (expert vs. apprentice). IgG levels against seven P. falciparum antigens with heterogeneous immunogenicities were measured in test samples, in a positive control and in blanks. We assessed the variability and MFI quantification range associated to each combination of conditions, and their interactions, and evaluated the minimum number of samples and blank replicates to achieve good replicability. Results showed that antigen immunogenicity and sample seroreactivity defined the optimal dilution to assess the effect of assay conditions on variability. We found that a unique antigen-bead coupling, samples prediluted daily, incubation at 22°C, and automatic washing, had lower variability. However, variability increased when performing several couplings and incubating at 22°C vs. 37°C. In addition, no effect of temperature was seen with a unique coupling. The expertise of the operator had no effect on assay variability but reduced the MFI quantification range. Finally, differences between sample replicates were minimal, and two blanks were sufficient to capture assay variability, as suggested by the constant Intraclass Correlation Coefficient of three and two blanks. To conclude, a single coupling was the variable that most consistently reduced assay variability, being clearly advisable. In addition, we suggest having more sample dilutions instead of replicates to increase the likelihood of sample MFIs falling in the linear part of the antigen-specific curve, thus increasing precision.

Dataset Information

Development of quantitative suspension array assays for six immunoglobulin isotypes and subclasses to multiple Plasmodium falciparum antigens.

Publications

Development of quantitative suspension array assays for six immunoglobulin isotypes and subclasses to multiple Plasmodium falciparum antigens.

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