Comparison of non-magnetic and magnetic beads multiplex assay for assessment of <i>Plasmodium falciparum</i> antibodies

Ondigo, Bartholomew N.; Park, Gregory S.; Ayieko, Cyrus; Nyangahu, Donald D.; Wasswa, Ronald; John, Chandy C.

doi:10.7717/peerj.6120

Cited by 11 publications

(18 citation statements)

References 21 publications

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“…For most proteins the coupled beads were highly stable (11/16 tested over 9-months), with the MFI dropping for three proteins and increasing for two proteins. This is supported by previous research that has indicated the stability of protein-coupled beads [13], noting that the stability may vary by antigen [15].…”

supporting

confidence: 75%

“…Comparison falciparum proteins [13]. This is despite different amounts of each protein being coupled to nonmagnetic versus magnetic beads ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…An important consideration is that up to 100 different proteins can be assayed simultaneously using nonmagnetic beads and a Bio-Plex ® 200 instrument, whereas the maximum is 50 proteins using a MAGPIX ® . If less than 50 proteins will be used, the MAGPIX ® instrument is cheaper and enables washing steps to be conducted with magnets, which improves both bead retention [13,16] and speed of the assay.…”

Section: Discussionmentioning

confidence: 99%

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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^®)

Mazhari

Brewster

Fong

et al. 2020

Preprint

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Multiplexed bead-based assays that use Luminex xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data is lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external validation of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were strongly correlated in all three of our comparisons, however higher amounts of protein were required for coupling to magnetic beads. Our external validation indicated that results generated in different laboratories using the same coupled beads are also highly comparable, particularly if a reference standard curve is used.

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supporting

confidence: 75%

“…Comparison falciparum proteins [13]. This is despite different amounts of each protein being coupled to nonmagnetic versus magnetic beads ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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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^®)

Mazhari

Brewster

Fong

et al. 2020

Preprint

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“…The overall aim of this study was to establish a BBMA with biotinylated SAG1 and SAG2A as antigens for analyzing seroconversion resulting from T. gondii infection in humans. Magnetic beads have distinct advantages over nonmagnetic beads, including the ease of processing and higher bead recovery [8,44]. Since at the beginning of these studies the Sav-coated MagPlex ® microbeads were not commercially available, we custom-prepared them by chemical coupling of Sav to various bead regions (see Methods).…”

Section: Expression Puri Cation and Characterization Of Biotinylatedmentioning

confidence: 99%

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays

Klein

Stern

Seeber

2020

Preprint

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BACKGROUND Few bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none are commercially available.RESULTS We report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1’s N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98-100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen. CONCLUSIONS Our recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii. They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. The described general expression strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies.

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Section: Bead-based Multiplex Assay With Biotinylated Sag1 and Sag2a mentioning

confidence: 99%

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays

Klein

Stern

Seeber

2020

Preprint

View full text Add to dashboard Cite

BACKGROUND Few bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none are commercially available. RESULTS We report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1’s N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98-100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen. CONCLUSIONS Our recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii . They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. Our strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies.

show abstract

Comparison of non-magnetic and magnetic beads multiplex assay for assessment of Plasmodium falciparum antibodies

Cited by 11 publications

References 21 publications

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^®)

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^®)

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays

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Comparison of non-magnetic and magnetic beads multiplex assay for assessment of Plasmodium falciparum antibodies

Cited by 11 publications

References 21 publications

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®)

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®)

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays

Contact Info

Product

Resources

About

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^®)

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^®)