The
P. vivax CSP protein contains a variable central region that is composed of two nanopeptides that repeat in tandem and result in the two major molecular variants of the protein, VK210 and VK247 [
18]. Local and global variations have been reported in the distribution of the two variants [
19,
20] and also variation in vector susceptibility towards the two forms [
21]. One of the challenges in investigating the anti-CSP protein immunity in
P. vivax samples is the consumption of twice the volume required for a
P. falciparum assay, which may exhaust the homogenate before subsequent molecular tests can be performed. This is particularly pressing when sample needs to be analysed for mixed species infections (and thus three ELISAs that each require 50 µL homogenate need to be run). To economize sample usage an approach was tested where a single 50 µL volume was transferred from one ELISA reaction plate to the next (Additional file
1: Figure S2). In this approach, the
P. falciparum plate was coated with 5 µg/mL of the capture monoclonal antibody overnight at 4 °C. Plates were washed three times with PBS, followed by blocking. 50 µL of mosquito homogenate, obtained as described above, was incubated on the
P. falciparum plate for 2 h at RT. At the same time, the VK210 plate was coated with its monoclonal antibody for 30 min at RT and washed three times with PBS, followed by blocking. At the end of the 2 h the homogenate was transferred with multichannel pipettes to the VK210 plate and incubated at RT for 2 h. The
P. falciparum plate was washed three times and incubated with HRP-tagged monoclonal for 1 h at RT. At the same time, the VK247 plate was coated with the capture antibody for 30 min at RT that was followed by three times washing and subsequent blocking. Mosquito homogenate was transferred to VK247 plates and incubated for 2 h at RT. The VK210 plates were washed three times and incubated with HRP-tagged monoclonal antibody for 1 h at RT. The
P. falciparum plate was washed four times and 100 µL of TMB was incubated for 20 min followed by addition of 50 µL stop solution. The VK247 plates were washed three times and incubated with HRP-tagged monoclonal antibody for 1 h at RT. The VK210 and VK247 plates were subsequently washed four times with PBS and incubated with TMB at RT for 20 min and reaction was stopped using 50 µL of 0.2 M H
2SO
4. In all cases 150 μL of blocking buffer (5% skimmed milk) was used to block plates for 1 h at RT.
All ELISA plates included mosquitoes fed on negative blood and threefold serial dilutions of positive controls of the respective reaction. To test for cross-reactivity, each plate also contained positive controls of the other two targets. Absorbance was read for all plates at 450 nm to determine optical density values using iMark™ microplate absorbance reader (Bio-Rad).