Development and optimization of the bead suspension array

Detection monoclonal mouse IgG α-PfHRP2 (MyBioSource, San Diego, CA, USA) and monoclonal mouse IgG α-PAN-pLDH (AccessBio, Somerset, NJ, USA) were biotinylated using the EZ-Link Sulfo-NHS-Biotin Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions with minor modifications (see Additional file 1: Text S1).

Coupling of magnetic microspheres was performed similarly as described elsewhere [28]. Briefly, two MagPlex^® microspheres (Luminex Corp., Austin, TX, USA) with different spectral signatures selected for the detection of PfHRP2 and PAN-pLDH were washed with distilled water and activated with Sulfo-NHS (N-hydroxysulfosuccinimide) and EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA), both at 50 mg/mL, in activation buffer (100 mM Monobasic Sodium Phosphate, pH = 6.2). Microspheres were washed with 50 mM MES potassium salt (4-morpholineethane sulfonic acid, Sigma Aldrich, St Louis, MO, USA) pH 5.0 to a 10,000 beads/µl concentration, and covalently coupled with capture antibodies against PfHRP2 (MyBiSource, San Diego, CA, USA) and PAN-pLDH (PA-12, AccessBio, Somerset, NJ, USA), both at a concentration of 25 µg/ml. Beads were incubated on a rotatory shaker overnight at 4 °C and protected from light. Microspheres were blocked with PBS-BN (PBS with 1% BSA and 0.05% sodium azide (Sigma, Tres Cantos, Madrid, Spain), and resuspended in PBS-BN (from now on named assay buffer) to be quantified on a Guava Personal Cell Analysis desktop cytometer (Guava, Hayward, CA, USA) to determine the percentage recovery after the coupling procedure. Coupling validation was performed by incubating 50 µl of each bead suspension (2000 beads/well) with 50 µl α-mouse IgG-Biotin (goat anti-Mouse IgG-Biotin, Sigma Aldrich, St Louis, MO, USA) at 1:1000 dilution in a 96-well flat bottom plate for 2 h in gentle agitation. The plate was washed by pelleting microspheres using a magnetic separator (EMDMillipore, Burlington, MA, USA) and resuspended with wash buffer (0.05% Tween 20/PBS). Beads were incubated with 100 µl streptavidin-phycoerythrin (Sigma Aldrich, St. Louis, MO, USA) diluted 1:1000 in assay buffer for 30-min with gentle agitation in the dark. Finally, the beads were washed and resuspended in assay buffer, and the plate was read using the Luminex xMAP^® 100/200 analyser (Luminex Corp., Austin, TX, USA). A reading higher than 25,000 median fluorescence intensity (MFI) implied a successful coupling reaction. Coupled beads were stored multiplexed at a concentration of 1000 beads/µl/region at 4 °C and protected from light.

To optimize the coupling concentration of detection antibodies, a concentration range from 10 to 100 µg/ml of α-PfHRP2 and α-PAN-pLDH mAbs was conjugated to magnetic beads, and assayed against serially diluted recombinant PfHRP2 and pLDH and a selection of plasma samples from P. falciparum-positive individuals. The mAb concentration that provided the highest MFI values was selected as the optimal concentration.

Recombinant PfHRP2 protein type A from FCQ79 P. falciparum strain expressed in Escherichia coli (890015, Microcoat GmbH, Germany) was selected as PfHRP2 reference material. Antigen concentration after reconstitution was determined by ELISA (Malaria Ag CELISA, CeLLabs, Australia). Purified recombinant P. falciparum and P. vivax pLDH proteins expressed in insect cells (3001, ReliaTech GmbH, Germany) were used as reference material. The pLDH concentrations were measured in a previous study using a commercially available ELISA (QUALISA Malaria kit, Qualpro Diagnostics, India) [21]. Reference materials were used to prepare the standard curves for the bead suspension array, starting at concentrations of 50 ng/ml for PfHRP2 type A and at 1000 ng/ml for P. falciparum and P. vivax pLDH. WHO International Standard for P. falciparum antigens was provided by the National Institute for Biological Standards and Control (Ridge, UK) (NIBSC code: 16/376). WHO International Standard for P. falciparum antigens was quantified, and the obtained antigen concentrations in pg/ml were used to calculate the number of antigen picograms corresponding to 1 International Unit (IU).

Standard curves were prepared for the detection of PfHRP2 and pLDH. The conjugated beads were incubated with serial dilutions of recombinant PfHRP2 types A, B and C and recombinant P. falciparum and P. vivax pLDH in assay buffer to produce standard curves ranging from 50,000 to 0.024 pg/ml for PfHRP2, and from 1000,000 to 0.48 for both P. falciparum pLDH and P. vivax pLDH (Fig. 1b) (for a more detailed assay procedure, see Additional file 1: Text S2).

Calibration curves to detect PfHRP2, Plasmodium falciparum pLDH and Plasmodium vivax pLDH. Recombinant P. falciparum HRP2 type A (a) and P. falciparum (b, back line) and P. vivax (B, grey line) pLDH were serially diluted to investigate the assay analytical range. Error bars show the standard deviation of the mean from 66 independent reads for PfHRP2 type A and P. falciparum pLDH, and 12 reads for P. vivax pLDH. X axis: MFI value after subtraction of the background