Analysis of factors affecting the variability of a quantitative suspension bead array assay measuring IgG to multiple Plasmodium antigens

Abstract

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.

Baseline exposure, antibody subclass, and hepatitis B response differentially affect malaria protective immunity following RTS,S/AS01E vaccination in African children

“…Quantitative suspension array technology (qSAT) [ 24 , 25 ] was used to measure antibody responses to three CSP constructs (FL, NANP repeat, and C-term [residues 274 to 387: KNNQG...SSIGL] recombinant proteins from WRAIR) and HBsAg (Abcam). The qSAT assays applied the xMAP™ technology (Luminex Corp., TX) and were previously standardized and optimized to control for sources of variability [ 26 , 27 ]. The multiplex antigen panel also included 32 P. falciparum proteins [ 26 , 27 ] analyzed as markers of malaria exposure and maternally transferred antibodies (see below).…”

“…The qSAT assays applied the xMAP™ technology (Luminex Corp., TX) and were previously standardized and optimized to control for sources of variability [ 26 , 27 ]. The multiplex antigen panel also included 32 P. falciparum proteins [ 26 , 27 ] analyzed as markers of malaria exposure and maternally transferred antibodies (see below). Briefly, antigen-coupled multiplex beads were mixed with 50 μL of test sample, negative or positive control [ 28 , 29 ] at multiple dilutions (see Additional file 1 : Supplementary methods).…”

Highly sensitive and specific multiplex antibody assays to quantify immunoglobulins M, A and G against SARS-CoV-2 antigens

“…Here we report on the establishment and validation of three quantitative suspension array technology (qSAT) assays to measure IgM, IgA and IgG antibodies against eight SARS-CoV-2 antigens, based on the adaptation of previous in-house protocols that measured antibodies to other infectious diseases, including malaria (15)(16)(17) (18). Due to the need to process a large amount of samples with the minimal time and cost during a pandemic like the COVID-19, we optimized several conditions to reduce the duration of the assays and report them here for the three main isotypes that have proved useful for seroprevalence studies (19).…”

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies againstP. vivaxantigens in a multiplexed bead-based assay using Luminex®technology (Bio-Plex®200 or MAGPIX®)

“…Proteins of interest can be coupled to a unique set of beads, facilitating multiplexed detection of antibody responses to multiple proteins. Several studies have been conducted with a focus on optimizing various steps of the coupling process or assay work-flow, in the context of detection of antibodies against Plasmodium proteins, such as bead coupling [4], sample pre-dilution [4], assay temperature [4], plate washing [4], operator expertise [4], incubation times [1], and bead numbers [5].…”